Compositions and methods for performing a stringent wash step in hybridization applications

ABSTRACT

The invention provides methods and compositions for performing a stringent wash step in a hybridization assay between at least one molecule and a target. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in the stringent wash step. Compositions for use in the invention include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary sequences in a hybridization product.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for performinga stringent wash step in hybridization applications In one embodiment,the present invention can be used for the in vivo, in vitro, and in situmolecular examination of DNA and RNA. In particular, the invention canbe used for the molecular examination of DNA and RNA in the fields ofcytology, histology, and molecular biology. In other embodiments, thepresent invention can be used for in situ hybridization (ISH)applications.

BACKGROUND AND DESCRIPTION

Double stranded nucleic acid molecules (i.e., DNA (deoxyribonucleicacid), DNA/RNA (ribonucleic acid) and RNA/RNA) associate in a doublehelical configuration. This double helix structure is stabilized byhydrogen bonding between bases on opposite strands when bases are pairedin a particular way (A+T/U or G+C) and hydrophobic bonding among thestacked bases. Complementary base paring (hybridization) is central toall processes involving nucleic acid.

In a basic example of hybridization, nucleic acid probes or primers aredesigned to bind, or “hybridize,” with a target nucleic acid, forexample, DNA or RNA in a sample. One type of hybridization application,in situ hybridization (ISH), includes hybridization to a target in aspecimen wherein the specimen may be in vivo, in situ, or for example,fixed or adhered to a glass slide.

The efficiency and accuracy of nucleic acid hybridization assays mostlydepend on at least one of three major factors: a) denaturation (i.e.,separation of, e.g., two nucleic acid strands) conditions, b)renaturation (i.e., re-annealing of, e.g., two nucleic acid strands)conditions, and c) post-hybridization washing conditions.

Traditional hybridization experiments, such as ISH assays, use aformamide-containing solution to denature doubled stranded nucleic acid.Formamide disrupts base pairing by displacing loosely and uniformlybound hydrate molecules and by causing “formamidation” of theWatson-Crick binding sites. Thus, formamide has a destabilizing effecton double stranded nucleic acids and analogs.

Once the complementary strands of nucleic acid have been separated, a“renaturation” or “reannealing” step allows the primers or probes tobind to the target nucleic acid in the sample. This step is alsosometimes referred to as the “hybridization” step. The re-annealing stepis by far the most time-consuming aspect of traditional hybridizationapplications. See FIGS. 1 and 2 (presenting examples of traditionalhybridization times). In addition, the presence of formamide in ahybridization buffer can significantly prolong the renaturation time, ascompared to aqueous denaturation solutions without formamide.

After the complementary strands of nucleic acid have reannealed, anyunbound and mis-paired probe is removed by a series ofpost-hybridization washes. The specificity of the interaction betweenthe probe and the target is largely determined by stringency of thesepost-hybridization washes. Duplexes containing highly complementarysequences are more resistant to high-stringency conditions than duplexeswith low complementary. Thus, increased stringency conditions can beused to remove non-specific bonds between the probe and the targetnucleic acids.

Four main variables are typically adjusted to influence the stringencyof the post-hybridization washes:

-   -   1. Temperature (as temperature increases, non-perfect matches        between the probe and the target sequence will denature, i.e.,        separate, before more perfectly matched sequences).    -   2. Salt conditions (as salt concentration decreases, non-perfect        matches between the probe and the target sequence will denature,        i.e., separate, before more perfectly matched sequences).    -   3. Formamide concentration (as the amount of formamide        increases, non-perfect matches between the probe and the target        sequence will denature, i.e., separate, before more perfectly        matched sequences).    -   4. Time (as the wash time increases, non-perfect matches between        the probe and the target sequence will denature, i.e., separate,        before more perfectly matched sequences).

Other factors such as pH, rate of agitation, and number of washes willalso influence the stringency of the wash step. However, the use of hightemperatures and/or high formamide concentrations can have significantdrawbacks. For example, heat can be destructive to membranes, samplemorphology, and to the nucleic acid itself. Heat can lead tocomplications when small volumes are used, since evaporation of aqueousbuffers is difficult to control. In addition, formamide is a toxic,hazardous material, subject to strict regulations for use and waste.Furthermore, the use of a high concentration of formamide appears tocause morphological destruction of cellular, nuclear, and/or chromosomalstructure.

Thus, a need exists for overcoming the drawbacks associated with thetraditional post-hybridization washes of hybridization applications. Byaddressing this need, the present invention provides several potentialadvantages over prior art hybridization applications, such as increasedspecificity, lower background, lower wash temperatures, preservation ofsample morphology, and less toxic hybridization solvents.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compositions whichresult in at least one of the following advantages: increasedspecificity, lower background, lower evaporation of reagent,preservation of sample morphology, simpler procedure, faster procedure,easier automation, and safer reagents. One way in which the presentinvention achieves those objectives is by providing compositions andmethods for performing a stringent wash step in hybridizationapplications.

The compositions and methods of the invention are applicable to anyhybridization technique. The compositions and methods of the inventionare also applicable to any molecular system that hybridizes or bindsusing base pairing, such as, for example, DNA, RNA, PNA, LNA, andsynthetic and natural analogs thereof.

The method and compositions of the present invention may be used for thein vivo, in vitro, or in situ analysis of genomic DNA, chromosomes,chromosome fragments, genes, and chromosome aberrations such astranslocations, deletions, amplifications, insertions, mutations, orinversions associated with a normal condition or a disease. Further, themethods and compositions are useful for detection of infectious agentsas well as changes in levels of expression of RNA, e.g., mRNA and itscomplementary DNA (cDNA).

Other uses include the in vivo, in vitro, or in situ analysis ofmessenger RNA (mRNA), viral RNA, viral DNA, small interfering RNA(siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g., tRNAand rRNA), transfer messenger RNA (tmRNA), micro RNA (miRNA),piwi-interacting RNA (piRNA), long noncoding RNA, small nucleolar RNA(snoRNA), antisense RNA, double-stranded RNA (dsRNA), methylations andother base modifications, single nucleotide polymorphisms (SNPs), copynumber variations (CNVs), and nucleic acids labeled with, e.g.,radioisotopes, fluorescent molecules, biotin, digoxigenin (DIG), orantigens, alone or in combination with unlabeled nucleic acids.

The method and compositions of the present invention are useful for invivo, in vitro, or in situ analysis of nucleic acids using techniquessuch as northern blot, Southern blot, flow cytometry, autoradiography,fluorescence microscopy, chemiluminescence, immunohistochemistry,virtual karyotype, gene assay, DNA microarray (e.g., array comparativegenomic hybridization (array CGH)), gene expression profiling, Gene ID,Tiling array, gel electrophoresis, capillary electrophoresis, and insitu hybridizations such as FISH, SISH, CISH. The methods andcompositions of the invention may be used on in vitro and in vivosamples such as bone marrow smears, blood smears, paraffin embeddedtissue preparations, enzymatically dissociated tissue samples, bonemarrow, amniocytes, cytospin preparations, imprints, etc.

In one embodiment, the invention provides methods and compositions forperforming a stringent wash step in hybridization applications. Theinvention may, for example, eliminate the use of, or reduce thedependence on formamide from, e.g., 50% formamide in traditionalstringent wash buffers to 25%, 15%, 10%, 5%, 2%, 1% or 0% formamide inthe compositions and methods of the invention.

The methods and compositions of the invention may also increase thestringency of post-hybridization washes without the use of formamidesuch that the stringent wash can occur, e.g., at a lower temperaturethan with traditional non-formamide stringent wash buffers.

The invention may also allow for stringency washes at lower temperatures(e.g. 20-30° C. lower) than traditional hybridization applications. Forexample, instead of washing single locus probes at 73° C. in 1×SSC, 0.3%NP-40, the compositions and methods of the invention may allow suchprobes to be washed at 45° C., or 40° C., or 35° C., or even at roomtemperature. The invention may also allow the stringent wash to beperformed at the same or at a lower temperature than the hybridizationtemperature.

Thus, in some aspects, the present invention overcomes major toxicityand temperature problems associated with traditional hybridizationassays by allowing for easier automation, by lowering the stringent washtemperature of, e.g., single locus probes without using the toxicchemical formamide, and by providing stringent wash temperatures thatcause less harm to, e.g., biological samples (such as, virus, RNA, DNA,FFPE tissue sections, cryo sections, cytological preparations, etc.)and/or their potential carriers (e.g. nitrocellulose membranes,microarrays, etc).

One aspect of the invention is a composition or solution for use inpost-hybridization stringency washes. Compositions for use in theinvention include an aqueous composition comprising at least one polaraprotic solvent in an amount effective to denature non-complementarydouble-stranded nucleotide sequences. One way to test for whether theamount of polar aprotic solvent is effective to denaturenon-complementary sequences in a hybridization product is to determinewhether the polar aprotic solvent, when used in the methods andcompositions described herein, yields a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay.

Non-limiting examples of effective amounts of polar aprotic solventsinclude, e.g., about 1% to about 95% (v/v). In some embodiments, theconcentration of polar aprotic solvent is 5% to 60% (v/v). In otherembodiments, the concentration of polar aprotic solvent is 10% to 60%(v/v). In still other embodiments, the concentration of polar aproticsolvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%,10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 60% to70% (v/v) are also suitable. In some embodiments, the polar aproticsolvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%,3%, 4%, or 5% (v/v). In other embodiments, the polar aprotic solventwill be present at a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%,10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%,16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).

According to another aspect of the present invention the aqueouscomposition comprising a polar aprotic solvent has reduced toxicitycompared to traditional formamide-containing stringency wash solutions.For example, a less-toxic composition of the invention may comprise acomposition with the proviso that the composition does not containformamide, or with the proviso that the composition contains less than25%, or less than 10%, or less than 5%, or less than 2%, or less than1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or lessthan 0.01% formamide. A less-toxic composition may also comprise acomposition with the proviso that the composition does not containdimethyl sulfoxide (DMSO), or with the proviso that the compositioncontains less than 25%, 10%, 5%, 2%, or less than 1%, or less than 0.5%,or less than 0.1%, or less than 0.05%, or less than 0.01% DMSO.

In one aspect of the invention, suitable polar aprotic solvents for usein the invention may be selected based on their Hansen SolubilityParameters. For example, suitable polar aprotic solvents may have adispersion solubility parameter between 17.7 to 22.0 MPa^(1/2), a polarsolubility parameter between 13 to 23 MPa^(1/2), and a hydrogen bondingsolubility parameter between 3 to 13 MPa^(1/2).

According to one aspect of the present invention, suitable polar aproticsolvents for use in the invention are cyclic compounds. A cycliccompound has a cyclic base structure.

Examples include the cyclic compounds disclosed herein. In otherembodiments, the polar aprotic solvent may be chosen from Formulas 1-4below:

where X is O and R1 is alkyldiyl.

According to another aspect of the invention, suitable polar aproticsolvents for use in the invention may be chosen from Formula 5 below:

where X is optional and if present, is chosen from O or S;where Z is optional and if present, is chosen from O or S;where A and B independently are O or N or S or part of the alkyldiyl ora primary amine;where R is alkyldiyl; andwhere Y is O or S or C.

Examples of suitable polar aprotic solvents according to Formula 5 areprovided in Formulas 6-9 below:

According to yet another aspect of the invention the polar aproticsolvent has lactone, sulfone, nitrile, sulfite, or carbonatefunctionality. Such compounds are distinguished by their relatively highdielectric constants, high dipole moments, and solubility in water.

According to another aspect of the invention the polar aprotic solventhaving lactone functionality is γ-butyrolactone (GBL), the polar aproticsolvent having sulfone functionality is sulfolane (SL), the polaraprotic solvent having nitrile functionality is acetonitrile (AN), thepolar aprotic solvent having sulfite functionality is glycolsulfite/ethylene sulfite (GS), and the polar aprotic solvent havingcarbonate functionality is ethylene carbonate (EC), propylene carbonate(PC), or ethylene thiocarbonate (ETC). In yet another aspect of theinvention, the compositions and methods of the invention comprise apolar aprotic solvent, with the proviso that the polar aprotic solventis not acetonitrile (AN) or sulfolane (SL).

According to yet another aspect, the invention discloses a method forperforming a stringent wash step in a hybridization applicationcomprising:

-   -   a) providing a hybridization product comprising a first nucleic        acid sequence hybridized to a second nucleic acid sequence,    -   b) providing an aqueous composition comprising at least one        polar aprotic solvent in an amount effective to denature        non-complementary double-stranded nucleotide sequences, and    -   c) combining the hybridization product and the aqueous        composition for at least a time period sufficient to denature        any non-complementary binding between the first and second        nucleic acid sequences.

In one embodiment, no additional energy is required to denature anynon-complementary binding between the first and second nucleic acidsequences. In other embodiments, a sufficient amount of energy todenature any non-complementary binding between the first and secondnucleic acid sequences is provided.

According to yet another aspect of the present invention, the energy isprovided by heating the aqueous composition and the hybridizationproduct. Thus, the step of method of the invention may include the stepsof heating and cooling the aqueous composition and nucleic acidsequences.

A further aspect of the invention, the energy is provided by the use ofmicrowaves, hot baths, hot plates, heat wire, peltier element, inductionheating, or heat lamps.

In one embodiment, the aqueous composition and hybridization product areheated to less than 70° C., such as, for example, 65° C., 62° C., 60°C., 55° C., 52° C., 50° C., 45° C., 42° C., or 40° C.

In one embodiment, the denaturation of any non-complementary bindingbetween the first and second nucleic acid sequences occurs in less than1 hour, such as, for example, 45 minutes, 30 minutes, 15 minutes, 10minutes, 5 minutes, or 1 minute.

According to a further aspect, the invention relates to the use of acomposition comprising between 1 and 95% (v/v) of at least one polaraprotic solvent in a stringent wash step in a hybridization application.

According to yet another aspect, the invention relates to the use of aan aqueous composition as described in this invention for performing astringent wash step in a hybridization application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a typical time-course for single locus hybridizationassay with primary labeled FISH probes on formaldehyde fixed paraffinembedded tissue sections (histological specimens). The first bar on theleft represents the deparaffination step; the second bar represents theheat-pretreatment step; the third bar represents the digestion step; thefourth bar represents the denaturation and hybridization steps; thefifth bar represents the stringency wash step; and the sixth barrepresents the mounting step.

FIG. 2 depicts a typical time-course for single locus hybridizationassay with primary labeled FISH probes on cytological specimens. Thefirst bar on the left represents the fixation step; the second barrepresents the denaturation and hybridization steps; the third barrepresents the stringency wash step; and the fourth bar represents themounting step.

DETAILED DESCRIPTION A. Definitions

In the context of the present invention the following terms are to beunderstood as follows:

“Biological sample” is to be understood as any in vivo, in vitro, or insitu sample of one or more cells or cell fragments. This can, forexample, be a unicellular or multicellular organism, tissue section,cytological sample, chromosome spread, purified nucleic acid sequences,artificially made nucleic acid sequences made by, e.g., a biologic basedsystem or by chemical synthesis, microarray, or other form of nucleicacid chip. In one embodiment, a sample is a mammalian sample, such as,e.g., a human, murine, rat, feline, or canine sample.

“Nucleic acid,” “nucleic acid chain,” and “nucleic acid sequence” meananything that binds or hybridizes using base pairing including,oligomers or polymers having a backbone formed from naturally occurringnucleotides and/or nucleic acid analogs comprising nonstandardnucleobases and/or nonstandard backbones (e.g., a peptide nucleic acid(PNA) or locked nucleic acid (LNA)), or any derivatized form of anucleic acid.

As used herein, the term “peptide nucleic acid” or “PNA” means asynthetic polymer having a polyamide backbone with pendant nucleobases(naturally occurring and modified), including, but not limited to, anyof the oligomer or polymer segments referred to or claimed as peptidenucleic acids in, e.g., U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049,5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461,5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470 6,201,103,6,228,982 and 6,357,163, WO96/04000, all of which are hereinincorporated by reference, or any of the references cited therein. Thependant nucleobase, such as, e.g., a purine or pyrimidine base on PNAmay be connected to the backbone via a linker such as, e.g., one of thelinkers taught in PCT/US02/30573 or any of the references cited therein.In one embodiment, the PNA has an N-(2-aminoethyl)-glycine) backbone.PNAs may be synthesized (and optionally labeled) as taught inPCT/US02/30573 or any of the references cited therein. PNAs hybridizetightly, and with high sequence specificity, with DNA and RNA, becausethe PNA backbone is uncharged. Thus, short PNA probes may exhibitcomparable specificity to longer DNA or RNA probes. PNA probes may alsoshow greater specificity in binding to complementary DNA or RNA.

As used herein, the term “locked nucleic acid” or “LNA” means anoligomer or polymer comprising at least one or more LNA subunits. Asused herein, the term “LNA subunit” means a ribonucleotide containing amethylene bridge that connects the 2′-oxygen of the ribose with the4′-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).

Examples of nucleic acids and nucleic acid analogs also include polymersof nucleotide monomers, including double and single strandeddeoxyribonucleotides (DNA), ribonucleotides (RNA), α-anomeric formsthereof, synthetic and natural analogs thereof, and the like. Thenucleic acid chain may be composed entirely of deoxyribonucleotides,ribonucleotides, peptide nucleic acids (PNA), locked nucleic acids(LNA), synthetic or natural analogs thereof, or mixtures thereof. DNA,RNA, or other nucleic acids as defined herein can be used in the methodand compositions of the invention.

“Polar aprotic solvent” refers to an organic solvent having a dipolemoment of about 2 debye units or more, a water solubility of at leastabout 5% (volume) at or near ambient temperature, i.e., about 20° C.,and which does not undergo significant hydrogen exchange atapproximately neutral pH, i.e., in the range of 5 to 9, or in the range6 to 8. Polar aprotic solvents include those defined according to theHansen Solubility Parameters discussed below.

“Alkyldiyl” refers to a saturated or unsaturated, branched, straightchain or cyclic hydrocarbon radical having two monovalent radicalcenters derived by the removal of one hydrogen atom from each of twodifferent carbon atoms of a parent alkane, alkene, or alkyne.

“Aqueous solution” is to be understood as a solution containing water,even small amounts of water. For example, a solution containing 1% wateris to be understood as an aqueous solution.

“Hybridization application,” “hybridization assay,” “hybridizationexperiment,” “hybridization procedure,” “hybridization technique,”“hybridization method,” etc. are to be understood as referring to anyprocess that involves hybridization of nucleic acids. Such a process mayinclude, for example, a fixation step, a deparaffination step, aheat-pretreatment step, a digestion step, denaturation and hybridizationsteps, a stringency wash step, and a mounting step. Unless otherwisespecified, the terms “hybridization” and “hybridization step” are to beunderstood as referring to the re-annealing step of the hybridizationprocedure as well as the denaturation step.

“Hybridization composition” refers to an aqueous solution of theinvention for performing a hybridization procedure, for example, to binda probe to a nucleic acid sequence. Hybridization compositions maycomprise, e.g., at least one polar aprotic solvent, at least one nucleicacid sequence, and a hybridization solution. Hybridization compositionsdo not comprise enzymes or other components, such as deoxynucleosidetriphosphates (dNTPs), for amplifying nucleic acids in a biologicalsample.

“Hybridization solution” refers to an aqueous solution for use in ahybridization composition of the invention. Hybridization solutions arediscussed in detail below and may comprise, e.g., buffering agents,accelerating agents, chelating agents, salts, detergents, and blockingagents.

“Hansen Solubility Parameters” and “HSP” refer to the following cohesionenergy (solubility) parameters: (1) the dispersion solubility parameter(δ_(D), “D parameter”), which measures nonpolar interactions derivedfrom atomic forces; (2) the polar solubility parameter (δ_(P), “Pparameter”), which measures permanent dipole-permanent dipoleinteractions; and (3) the hydrogen bonding solubility parameter (δ_(H),“H parameter”), which measures electron exchange. The Hansen SolubilityParameters are further defined below.

“Repetitive Sequences” is to be understood as referring to the rapidlyreannealing (approximately 25%) and/or intermediately reannealing(approximately 30%) components of mammalian genomes. The rapidlyreannealing components contain small (a few nucleotides long) highlyrepetitive sequences usually found in tandem (e.g., satellite DNA),while the intermediately reannealing components contain interspersedrepetitive DNA. Interspersed repeated sequences are classified as eitherSINEs (short interspersed repeat sequences) or LINEs (long interspersedrepeated sequences), both of which are classified as retrotransposons inprimates. SINEs and LINEs include, but are not limited to, Alu-repeats,Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,tetra-nucleotide repeats, penta-nucleotide repeats and hexa-nucleotiderepeats. Alu repeats make up the majority of human SINEs and arecharacterized by a consensus sequence of approximately 280 to 300 bpthat consist of two similar sequences arranged as a head to tail dimer.In addition to SINEs and LINEs, repeat sequences also exist inchromosome telomeres at the termini of chromosomes and chromosomecentromeres, which contain distinct repeat sequences that exist only inthe central region of a chromosome. However, unlike SINEs and LINEs,which are dispersed randomly throughout the entire genome, telomere andcentromere repeat sequences are localized within a certain region of thechromosome.

“Non-toxic” and “reduced toxicity” are defined with respect to thetoxicity labeling of formamide according to “Directive 1999/45/EC of theEuropean Parliament and of the Council of 31 May 1999 concerning theapproximation of the laws, regulations and administrative provisions ofthe Member States relating to the classification, packaging, andlabelling of dangerous preparations”(ecb.jrc.it/legislation/1999L0045EC.pdf) (“Directive”). According to theDirective, toxicity is defined using the following classification order:T+ “very toxic”; T “toxic”, C “corrosive”, Xn “harmful”, Xi “irritant.”Risk Phrases (“R phrases”) describe the risks of the classifiedtoxicity. Formamide is listed as T (toxic) and R61 (may cause harm tothe unborn child). All of the following chemicals are classified as lesstoxic than formamide: acetonitrile (Xn, R11, R20, R21, R22, R36);sulfolane (Xn, R22); γ-butyrolactone (Xn, R22, R32); and ethylenecarbonate (Xi, R36, R37, R38). At the time of filing this application,ethylene trithiocarbonate and glycol sulfite are not presently labeled.

“Stringent” and “stringency” in the context of post-hybridization washesare to be understood as referring to conditions for denaturingnon-complementary base-pairing. In general, a signal to noise ratio of2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates specific hybridization withlittle non-complementary base-pairing.

Stringent wash conditions are sequence dependent and are different underdifferent environmental parameters. The melting temperature (T_(m)) canbe used to adjust wash conditions to increase complementary basepairing. T_(m) is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. For DNA-DNA hybrids, the T_(m) can be approximated from thefollowing equation:

T _(m)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L

where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching. Accordingly, stringent conditions are generally selected tobe about 5° C. lower than T_(m) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a wash at 1, 2, 3, or 4° C. lower thanT_(m); moderately stringent conditions can utilize a wash at 6, 7, 8, 9,or 10° C. lower than T_(m); low stringency conditions can utilize a washat 11, 12, 13, 14, 15, or 20° C. lower than T_(m). Using the equation,those of ordinary skill will understand that variations in thestringency of wash solutions are inherently described.

As used herein, the terms “room temperature” and “RT” refer to about 20°C. to about 25° C., unless otherwise stated.

B. Solvent Selection

Suitable polar aprotic solvents for use in the invention may be selectedbased on their Hansen Solubility Parameters. Methods for experimentallydetermining and/or calculating HSP for a solvent are known in the art,and HSP have been reported for over 1200 chemicals.

For example, the D parameter may be calculated with reasonable accuracybased on refractive index, or may be derived from charts by comparisonwith known solvents of similar size, shape, and composition afterestablishing a critical temperature and molar volume. The P parametermay be estimated from known dipole moments (see, e.g., McClellan A. L.,Tables of Experimental Dipole Moments (W.H. Freeman 1963)) usingEquation 1:

δ_(P)=37.4(Dipole Moment)/V ^(1/2)  Equation 1

where V is the molar volume. There are no equations for calculating theH parameter. Instead, the H parameter is usually determined based ongroup contributions. HSP characterizations are conveniently visualizedusing a spherical representation, with the HSP of anexperimentally-determined suitable reference solvent at the center ofthe sphere. The radius of the sphere (R) indicates the maximum tolerablevariation from the HSP of the reference solvent that still allows for a“good” interaction to take place. Good solvents are within the sphereand bad ones are outside. The distance, R_(a), between two solventsbased on their respective HSP values can be determined using Equation 2:

(R _(a))²=4(δ_(D1)−δ_(D2))²+(δ_(P1)−δ_(P2))²(δ_(H1)−δ_(H2))²  Equation 2

where subscript 1 indicates the reference sample, subscript 2 indicatesthe test chemical, and all values are in MPa^(1/2). Good solubilityrequires that R_(a) be less than the experimentally-determined radius ofthe solubility sphere R_(o). The relative energy difference between twosolvents, i.e., RED number, can be calculated by taking the ratio ofR_(a) to R_(o), as shown in Equation 3.

RED=R _(a) /R _(o)  Equation 3

RED numbers less than 1.0 indicate high affinity; RED numbers equal orclose to 1.0 indicate boundary conditions; and progressively higher REDnumbers indicate progressively lower affinities.

In some embodiments, the D parameters of the polar aprotic solvents ofthe invention are between 17.7 to 22.0 MPa^(1/2). Such relatively high Dparameters are generally associated with solvents having cyclicstructures and/or structures with sulfur or halogens. Linear compoundsare not likely to be among the most suitable polar aprotic solvents foruse in the invention, but may be considered if their P and H parametersare within the ranges discussed below. Since the D parameter ismultiplied by 4 in Equation 2, the limits are one-half of R_(o). Inaddition, it should be noted that D values of around 21 or higher areoften characteristic of a solid.

In some embodiments, the P parameters of the polar aprotic solvents ofthe invention are between 13 to 23 MPa^(1/2). Such exceptionally high Pparameters are generally associated with solvents having a high dipolemoment and presumably also a relatively low molecular volume. Forexample, for V near 60 cc/mole, the dipole moment should be between 4.5and 3.1. For V near 90 cc/mole, the dipole moment should be between 5.6and 3.9.

In some embodiments, the H parameters of the polar aprotic solvents ofthe invention are between 3 to 13 MPa^(1/2). Generally, polar aproticsolvents having an alcohol group are not useful in the compositions andmethods of the invention, since the H parameters of such solvents wouldbe too high.

The molar volume of the polar aprotic solvent may also be relevant,since it enters into the evaluation of all three Hansen SolubilityParameters. As molar volume gets smaller, liquids tend to evaporaterapidly. As molar volume gets larger, liquids tend to enter the solidregion in the range of D and P parameters recited above. Thus, the polaraprotic solvents of the invention are rather close to the liquid/solidboundary in HSP space.

In some embodiments, the polar aprotic solvents of the invention havelactone, sulfone, nitrile, sulfite, and/or carbonate functionality. Suchcompounds are distinguished by their relatively high dielectricconstants, high dipole moments, and solubility in water. An exemplarypolar aprotic solvent with lactone functionality is γ-butyrolactone(GBL), an exemplary polar aprotic solvent with sulfone functionality issulfolane (SL; tetramethylene sulfide-dioxide), an exemplary polaraprotic solvent with nitrile functionality is acetonitrile (AN), anexemplary polar aprotic solvent with sulfite functionality is glycolsulfite/ethylene sulfite (GS), and an exemplary polar aprotic solventswith carbonate functionality are ethylene carbonate (EC), propylenecarbonate (PC), or ethylene trithiocarbonate (ETC). The structures ofthese exemplary solvents are provided below and their Hansen SolubilityParameters, RED numbers, and molar volumes are given in Table 1.

TABLE 1 Molar Volume D P H RED (cm³/mole) Correlation  19.57  19.11 7.71 — — (R₀ = 3.9) GBL 19.0 16.6 7.4 0.712 76.5 PC 20.0 18.0 4.1 0.99385.2 SL 20.3 18.2 10.9  0.929 95.7 EC 19.4 21.7 5.1 0.946 66.0 ETC n/an/a n/a n/a n/a GS 20.0 15.9 5.1 n/a 75.1 n/a = not available.

Other suitable polar aprotic solvents that may be used in the inventionare cyclic compounds such as, e.g., ∈-caprolactone. In addition,substituted pyrrolidinones and related structures with nitrogen in a 5-or 6-membered ring, and cyclic structures with two nitrile groups, orone bromine and one nitrile group, may also be suitable for use in theinvention. For example, N-methylpyrrolidinone (shown below) may be asuitable polar aprotic solvent for use in the methods and compositionsof the invention.

Other suitable polar aprotic solvents may contain a ring urethane group(NHCOO—). However, not all such compounds are suitable. One of skill inthe art may screen for compounds useful in the compositions and methodsof the invention as described herein. Exemplary chemicals that may besuitable for use in the invention are set forth in Tables 2 and 3 below.

TABLE 2 Solvent D P H Acetanilide 20.6 13.3 12.4 N-Acetyl Pyrrolidone17.8 13.1 8.3 4-Amino Pyridine 20.4 16.1 12.9 Benzamide 21.2 14.7 11.2Benzimidazole 20.6 14.9 11.0 1,2,3-Benzotriazole 18.7 15.6 12.4Butadienedioxide 18.3 14.4 6.2 2,3-Butylene Carbonate 18.0 16.8 3.1Caprolactone (Epsilon) 19.7 15.0 7.4 Chloro Maleic Anhydride 20.4 17.311.5 2-Chlorocyclohexanone 18.5 13.0 5.1 Chloronitromethane 17.4 13.55.5 Citraconic Anhydride 19.2 17.0 11.2 Crotonlactone 19.0 19.8 9.6Cyclopropylnitrile 18.6 16.2 5.7 Dimethyl Sulfate 17.7 17.0 9.7 DimethylSulfone 19.0 19.4 12.3 Dimethyl Sulfoxide 18.4 16.4 10.21,2-Dinitrobenzene 20.6 22.7 5.4 2,4-Dinitrotoluene 20.0 13.1 4.9Dipheynyl Sulfone 21.1 14.4 3.4 1,2-Dinitrobenzene 20.6 22.7 5.42,4-Dinitrotoluene 20.0 13.1 4.9 Epsilon-Caprolactam 19.4 13.8 3.9Ethanesulfonylchloride 17.7 14.9 6.8 Furfural 18.6 14.9 5.12-Furonitrile 18.4 15.0 8.2 Isoxazole 18.8 13.4 11.2 Maleic Anhydride20.2 18.1 12.6 Malononitrile 17.7 18.4 6.7 4-Methoxy Benzonitrile 19.416.7 5.4 1-Methoxy-2-Nitrobenzene 19.6 16.3 5.5 1-Methyl Imidazole 19.715.6 11.2 3-Methyl Isoxazole 19.4 14.8 11.8 N-Methyl Morpholine-N- 19.016.1 10.2 Oxide Methyl Phenyl Sulfone 20.0 16.9 7.8 Methyl Sulfolane19.4 17.4 5.3 Methyl-4-Toluenesulfonate 19.6 15.3 3.8 3-Nitroaniline21.2 18.7 10.3 2-Nitrothiophene 19.7 16.2 8.2 9,10-Phenanthrenequinone20.3 17.1 4.8 Phthalic Anhydride 20.6 20.1 10.1 1,3-Propane Sultone 18.416.0 9.0 beta-Propiolactone 19.7 18.2 10.3 2-Pyrrolidone 19.4 17.4 11.3Saccharin 21.0 13.9 8.8 Succinonitrile 17.9 16.2 7.9 Sulfanilamide 20.019.5 10.7 Sulfolane 20.3 18.2 10.9 2,2,6,6- 19.5 14.0 6.3Tetrachlorocyclohexanone Thiazole 20.5 18.8 10.8 3,3,3-Trichloro Propene17.7 15.5 3.4 1,1,2-Trichloro Propene 17.7 15.7 3.4 1,2,3-TrichloroPropene 17.8 15.7 3.4

Table 2 sets forth an exemplary list of potential chemicals for use inthe compositions and methods of the invention based on their HansenSolubility Parameters. Other compounds, may of course, also meet theserequirements such as, for example, those set forth in Table 3.

TABLE 3 Chemical (dipole moment) RED Melting Point ° C. Chloroethylenecarbonate (4.02) 0.92 — 2-Oxazolidinone (5.07) 0.48 86-89 2-Imidazole1.49 90-91 1,5-Dimethyl Tetrazole (5.3) ~1.5   70-72 N-Ethyl Tetrazole(5.46) ~1.5   Trimethylene sulfide-dioxide (4.49) — — Trimethylenesulfite (3.63) — — 1,3-Dimethyl-5-Tetrazole (4.02) — — Pyridazine (3.97)1.16 −8 2-Thiouracil (4.21) — — N-Methyl Imidazole (6.2) 1.28 —1-Nitroso-2-pyrolidinone ~1.37   — Ethyl Ethyl Phosphinate (3.51) — —5-cyano-2-Thiouracil (5.19) — — 4H-Pyran-4-thione (4.08) 1.35 32-344H-Pyran-4-one = gamma pyrone (4.08) 1.49 Boiling Point (BP) 802-Nitrofuran (4.41) 1.14 29 Methyl alpha Bromo Tetronate (6.24) — —Tetrahydrothiapyran oxide (4.19) 1.75 60-64 Picolinonitrile(2-cyanopyridine) (5.23) 0.40 26-28 (BP 212-215) Nitrobenzimidazole(6.0) 0.52 207-209 Isatin (5.76) — 193-195 N-phenyl sydnone (6.55) — —Glycol sulfate (Ethylene glycol) — 99° C. Note: not soluble at 40%

Some of the chemicals listed in Tables 2 and 3 have been used inhybridization and/or PCR applications in the prior art (e.g., dimethylsulfoxide (DMSO) has been used in hybridization and PCR applications,and sulfolane (SL), acetonitrile (AN), 2-pyrrolidone, ∈-caprolactam, andethylene glycol have been used in PCR applications). Thus, in someembodiments, the polar aprotic solvent is not DMSO, sulfolane,acetonitrile, 2-pyrrolidone, ∈-caprolactam, or ethylene glycol. However,most polar aprotic solvents have not been used in prior arthybridization applications. Moreover, even when such compounds wereused, the prior art did not recognize that they may be advantageouslyused in the stringent wash step of such hybridization applications, asdisclosed in this application.

In addition, not all of the chemicals listed in Tables 2 and 3 aresuitable for use in the compositions and methods of the invention. Forexample, although DMSO is listed in Table 2 because its HansenSolubility Parameters (HSPs) fall within the ranges recited above, DMSOlikely does not function in the stringent wash compositions and methodsof the invention. However, it is well within the skill of the ordinaryartisan to screen for suitable compounds using the guidance providedherein including testing a compound in one of the examples provided. Forexample, in some embodiments, suitable polar aprotic solvents will haveHSPs within the ranges recited above and a structure shown in Formulas1-9 above.

C. Compositions, Buffers, and Solutions

(1) Stringent Wash Solutions

Traditional stringent wash solutions are known in the art. Suchsolutions may comprise, for example, buffering agents, acceleratingagents, chelating agents, salts, detergents, and blocking agents.

For example, the buffering agents may include SSC, HEPES, SSPE, PIPES,TMAC, TRIS, SET, citric acid, a phosphate buffer, such as, e.g.,potassium phosphate or sodium pyrrophosphate, etc. The buffering agentsmay be present at concentrations from 0.01× to 50×, such as, forexample, 0.01×, 0.1×, 0.5×, 1×, 2×, 5×, 10×, 15×, 20×, 25×, 30×, 35×,40×, 45×, or 50×. Typically, the buffering agents are present atconcentrations from 0.1× to 10×.

The accelerating agents may include polymers such as FICOLL, PVP,heparin, dextran sulfate, proteins such as BSA, glycols such as ethyleneglycol, glycerol, 1,3 propanediol, propylene glycol, or diethyleneglycol, combinations thereof such as Dernhardt's solution and BLOTTO,and organic solvents such as formamide, dimethylformamide, DMSO, etc.The accelerating agent may be present at concentrations from 1% to 80%or 0.1× to 10×, such as, for example, 0.1% (or 0.1×), 0.2% (or 0.2×),0.5% (or 0.5×), 1% (or 1×), 2% (or 2×), 5% (or 5×), 10% (or 10×), 15%(or 15×), 20% (or 20×), 25% (or 25×), 30% (or 30×), 40% (or 40×), 50%(or 50×), 60% (or 60×), 70% (or 70×), or 80% (or 80×). Typically,formamide is present at concentrations from 25% to 75%, such as 25%,30%, 40%, 50%, 60%, 70%, or 75%, while DMSO, dextran sulfate, and glycolare present at concentrations from 5% to 10%, such as 5%, 6%, 7%, 8%,9%, or 10%.

The chelating agents may include EDTA, EGTA, etc. The chelating agentsmay be present at concentrations from 0.1 mM to 10 mM, such as 0.1 mM,0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or10 mM. Typically, the chelating agents are present at concentrationsfrom 0.5 mM to 5 mM, such as 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM,3.5 mM, 4 mM, 4.5 mM, or 5 mM.

The salts may include sodium chloride, sodium phosphate, magnesiumphosphate, etc. The salts may be present at concentrations from 1 mM to750 mM, such as 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM,200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, or 750 mM. Typically,the salts are present at concentrations from 10 mM to 500 mM, such as 10mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500mM.

The detergents may include Tween, SDS, Triton, CHAPS, deoxycholic acid,etc. The detergent may be present at concentrations from 0.001% to 10%,such as, for example, 0.0001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10%. Typically, the detergents are present at concentrations from0.01% to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

A great variation exists in the literature regarding traditionalhybridization wash solutions. For instance, an example of a typicalstringent wash is 1×SSC (20×SSC=3M NaCl, 0.3M trisodium citrate, pH 7.0)at 65° C. for 10 minutes. Another example of a typical stringent washis: first a high stringency wash with 0.4× (or 1×) SSC, 0.3% NP-40, pH7.0 at 73° C. for 2 min., followed by a medium stringency wash with2×SSC, 0.1% NP-40, pH 7.0 at room temperature for 1-10 min. For example,a typical stringent wash for nucleic acids which have more than 100complementary residues is a 0.1× to 0.2×SSC at 60 to 65° C. for 15minutes. An example a typical medium stringency wash for nucleic acidswhich have more than 100 complementary residues is 0.5× to 1×SSC at 45to 55° C. for 15 minutes. An example a typical low stringency wash fornucleic acids which have more than 100 complementary residues is 2× to5×SSC at 40 to 50° C. for 15 minutes. For shorter probes (e.g., about 10to 50 nucleotides), stringent conditions typically involve saltconcentrations of less than about 1.5 M, more typically about 0.01 to1.0 M, at pH 7.0 to 8.3, and the temperature is typically at least about30° C.

Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For example, for posthybridization washes of single locus FISH with large DNA probes basedon, e.g., BAC and Cosmid clones hybridized at 37° C.: stringent washesmay use 2×SSC and 50% formamide for 3×5 min from 42 to 45° C. Inaddition, stringent conditions with formamide may be optimized fordifferent types and lengths of probes (e.g. DNA oligos; LNA; PNA) anddifferent targets (e.g. mRNA, virus).

The compositions of the invention may comprise a wash solutioncomprising any of the components of traditional components recited abovein combination with at least one polar aprotic solvent. The traditionalcomponents may be present at the same concentrations as used intraditional wash solutions, or may be present at higher or lowerconcentrations, or may be omitted completely.

For example, if the compositions of the invention comprise salts such asNaCl and/or phosphate buffer, the salts may be present at concentrationsof 0-1200 mM NaCl and/or 0-200 mM phosphate buffer. In some embodiments,the concentrations of salts may be, for example, 0 mM, 15 mM, 30 mM, 45mM, 60 mM, 75 mM, 90 mM, 105 mM, 120 mM, 135 mM, 150 mM, 165 mM, 180 mM,195 mM, 210 mM, 225 mM, 240 mM, 255 mM, 270 mM, 285 mM, or 300 mM NaCland 5 mM phosphate buffer, or 600 mM NaCl and 10 mM phosphate buffer. Inother embodiments, the concentrations of salts may be, for example, theconcentrations present in 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×,0.8×, 0.9×, 1×, 2×, 3×, 4×, 5×, 6×, 7×, or 8×SSC.

If the compositions of the invention comprise accelerating agents suchas dextran sulfate, glycol, or DMSO, the dextran sulfate may be presentat concentrations of from 5% to 40%, the glycol may be present atconcentrations of from 0.1% to 10%, and the DMSO may be from 0.1% to10%. In some embodiments, the concentration of dextran sulfate may be10% or 20% and the concentration of ethylene glycol, 1,3 propanediol, orglycerol may be 1% to 10%. In some embodiments, the concentration ofDMSO may be 1%. In some embodiments, the aqueous composition does notcomprise DMSO as an accelerating agent. In some embodiments, the aqueouscomposition does not comprise formamide as an accelerating agent, orcomprises formamide with the proviso that the composition contains lessthan 25%, or less than 10%, or less than 5%, or less than 2%, or lessthan 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, orless than 0.01%.

If the compositions of the invention comprise citric acid, theconcentrations may range from 1 mM to 100 mM and the pH may range from5.0 to 8.0. In some embodiments the concentration of citric acid may be10 mM and the pH may be 6.2.

The compositions of the invention may comprise agents that reducenon-specific binding to, for example, the cell membrane, such as salmonsperm or small amounts of total human DNA or, for example, they maycomprise blocking agents to block binding of, e.g., repeat sequences tothe target such as larger amounts of total human DNA or repeat enrichedDNA or specific blocking agents such as PNA or LNA fragments andsequences. These agents may be present at concentrations of from0.01-100 μg/μL or 0.01-100 μM. For example, in some embodiments, theseagents will be 0.1 μg/μL total human DNA, or 0.1 μg/μL non-human DNA,such as herring sperm, salmon sperm, or calf thymus DNA, or 5 μMblocking PNA.

One aspect of the invention is a composition or solution for use in astringent wash step in a hybridization application. Compositions for usein the invention include an aqueous composition comprising at least onepolar aprotic solvent in an amount effective to denaturenon-complementary double-stranded nucleotide sequences. One way to testfor whether the amount of polar aprotic solvent is effective to denaturenon-complementary sequences in a hybridization product is to determinewhether the polar aprotic solvent, when used in the methods andcompositions described herein, yields a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay.

Non-limiting examples of effective amounts of polar aprotic solventsinclude, e.g., about 1% to about 95% (v/v). In some embodiments, theconcentration of polar aprotic solvent is 5% to 60% (v/v). In otherembodiments, the concentration of polar aprotic solvent is 10% to 60%(v/v). In still other embodiments, the concentration of polar aproticsolvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%,10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% (v/v) arealso suitable. In some embodiments, the polar aprotic solvent will bepresent at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5%(v/v). In other embodiments, the polar aprotic solvent will be presentat a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%,11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%,17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).

In one embodiment, a composition of the invention comprises a mixture of20% polar aprotic solvent (v/v) (e.g., ethylene carbonate, “EC”) and2×SSC at pH 7.0. Another exemplary composition of the present inventioncomprises a mixture of 50% EC and 2×SSC at pH 7.0.

(2) Polar Aprotic Solvent(s)

Different polar aprotic solvents may impart different properties on thecompositions of the invention. For example, the choice of polar aproticsolvent may contribute to the stability of the composition, sincecertain polar aprotic solvents may degrade over time. For example, thepolar aprotic solvent ethylene carbonate breaks down into ethyleneglycol, which is a relatively stable molecule, and carbon dioxide, whichcan interact with water to form carbonic acid, altering the acidity ofthe compositions of the invention. Without being bound by theory, it isbelieved that the change in pH upon breakdown of ethylene carbonate andDNA damage from long storage makes the compositions of the inventionless effective. However, stability can be improved by reducing the pH ofthe composition, by adding citric acid as a buffer at pH 6.2 instead ofthe traditional phosphate buffer, which is typically used at about pH7.4, and/or by adding ethylene glycol at concentrations, e.g., between0.1% to 10%, or between 0.5% to 5%, such as, for example, 1%, 2%, 3%,etc. For example, with 10 mM citrate buffer, the compositions of theinvention are stable at 2-8° C. for approximately 8 months. Stabilitycan also be improved if the compositions are stored at low temperatures(e.g., −20° C.).

In addition, certain polar aprotic solvents may cause the compositionsof the invention to separate into multi-phase systems under certainconditions. The conditions under which multi-phase systems are obtainedmay be different for different polar aprotic solvents. Generally,however, as the concentration of polar aprotic solvent increases, thenumber of phases increases. For example, compositions comprising lowconcentrations ethylene carbonate (i.e., less than 20%) may exist as onephase, while compositions comprising higher concentrations of ethylenecarbonate may separate into two, or even three phases. For instance,compositions comprising 15% ethylene carbonate exist as a single phaseat room temperature, while compositions comprising 40% ethylenecarbonate consist of a viscous lower phase (approximately 25% of thetotal volume) and a less viscous upper phase (approximately 75% of thetotal volume) at room temperature. On the other hand, compositionscomprising greater than 20% EC, for example 40% or 50% EC, and 2×SSC maybe present as a single phase at room temperature.

On the other hand, some polar aprotic solvents may exist in two phasesat room temperature even at low concentrations. For example, sulfolane,γ-butyrolactone, ethylene trithiocarbonate, glycol sulfite, andpropylene carbonate exist as two phases at concentrations of 10, 15, 20,or 25% (20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer) at roomtemperature.

It may also be possible to alter the number of phases by adjusting thetemperature of the compositions of the invention. Generally, astemperature increases, the number of phases decreases. For example, at2-8° C., compositions comprising 40% ethylene carbonate may separateinto a three-phase system.

It may also be possible to alter the number of phases if present in thecompositions by adjusting the concentration of salt in the composition.Generally speaking, lowering the salt concentration may reduce thenumber of phases. However, depending on the particular polar aproticsolvent and its concentration in the composition, single phases may beproduced even with higher concentrations of salt.

Washes may be performed with a one-phase composition of the invention,with individual phases of the multiphase compositions of the invention,or with mixtures of any one or more of the phases in a multiphasecomposition of the invention.

(3) Optimization for Particular Applications

The compositions of the invention can be varied in order to optimizeresults for a particular application. For example, the concentration ofpolar aprotic solvent, salt, accelerating agent, blocking agent, and/orhydrogen ions (i.e. pH) may be varied in order to improve results for aparticular application. For example, the concentration of polar aproticsolvent may be varied in order to improve signal intensity andbackground staining.

D. Applications, Methods, and Uses

(1) Analytical Samples

The methods and compositions of the invention may be used fully orpartly in all types of hybridization applications in the fields ofcytology, histology, or molecular biology. According to one embodiment,the first or the second nucleic acid sequence in the methods of theinvention is present in a biological sample. Examples of such samplesinclude, e.g., tissue samples, cell preparations, cell fragmentpreparations, and isolated or enriched cell component preparations. Thesample may originate from various tissues such as, e.g., breast, lung,colorectal, prostate, lung, head & neck, stomach, pancreas, esophagus,liver, and bladder, or other relevant tissues and neoplasia thereof, anycell suspension, blood sample, fine needle aspiration, ascites fluid,sputum, peritoneum wash, lung wash, urine, feces, cell scrape, cellsmear, cytospin or cytoprep cells.

The sample may be isolated and processed using standard protocols. Cellfragment preparations may, e.g., be obtained by cell homogenizing,freeze-thaw treatment or cell lysing. The isolated sample may be treatedin many different ways depending of the purpose of obtaining the sampleand depending on the routine at the site. Often the sample is treatedwith various reagents to preserve the tissue for later sample analysis,alternatively the sample may be analyzed directly. Examples of widelyused methods for preserving samples are formalin-fixed followed byparaffin-embedding and cryo-preservation.

For metaphase spreads, cell cultures are generally treated withcolcemid, or anther suitable spindle pole disrupting agent, to stop thecell cycle in metaphase. The cells are then fixed and spotted ontomicroscope slides, treated with formaldehyde, washed, and dehydrated inethanol. Probes are then added and the samples are analyzed by any ofthe techniques discussed below.

Cytology involves the examination of individual cells and/or chromosomespreads from a biological sample. Cytological examination of a samplebegins with obtaining a specimen of cells, which can typically be doneby scraping, swabbing or brushing an area, as in the case of cervicalspecimens, or by collecting body fluids, such as those obtained from thechest cavity, bladder, or spinal column, or by fine needle aspiration orfine needle biopsy, as in the case of internal tumors. In a conventionalmanual cytological preparation, the sample is transferred to a liquidsuspending material and the cells in the fluid are then transferreddirectly or by centrifugation-based processing steps onto a glassmicroscope slide for viewing. In a typical automated cytologicalpreparation, a filter assembly is placed in the liquid suspension andthe filter assembly both disperses the cells and captures the cells onthe filter. The filter is then removed and placed in contact with amicroscope slide. The cells are then fixed on the microscope slidebefore analysis by any of the techniques discussed below.

In a traditional DNA hybridization experiment using a cytologicalsample, slides containing the specimen are immersed in a formaldehydebuffer, washed, and then dehydrated in ethanol. The probes are thenadded and the specimen is covered with a coverslip. The slide isoptionally incubated at a temperature sufficient to denature anydouble-stranded nucleic acid in the specimen (e.g., 5 minutes at 82° C.)and then incubated at a temperature sufficient to allow hybridization(e.g., overnight at 45° C.). After hybridization, the coverslips areremoved and the specimens are subjected to a high-stringency wash (e.g.,10 minutes at 65° C.) followed by a series of low-stringency washes(e.g., 2×3 minutes at room temperature). The samples are then dehydratedand mounted for analysis.

In a traditional RNA hybridization experiment using cytological samples,cells are equilibrated in 40% formamide, 1×SSC, and 10 mM sodiumphosphate for 5 min, incubated at 37° C. overnight in hybridizationreactions containing 20 ng of oligonucleotide probe (e.g mix of labeled50 bp oligos), 1×SSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20mM ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E. colitRNA (10 mg/ml), and 10 mM sodium phosphate. Then washed twice with4×SSC/40% formamide and again twice with 2×SSC/40% formamide, both at37° C., and then with 2×SSC three times at room temperature.Digoxigenin-labeled probes can then e.g. be detected by using amonoclonal antibody to digoxigenin conjugated to Cy3. Biotin-labeledprobes can then e.g. be detected by using streptavidin-Cy5. Detectioncan be by fluorescence or CISH.

Histology involves the examination of cells in thin slices of tissue. Toprepare a tissue sample for histological examination, pieces of thetissue are fixed in a suitable fixative, typically an aldehyde such asformaldehyde or glutaraldehyde, and then embedded in melted paraffinwax. The wax block containing the tissue sample is then cut on amicrotome to yield thin slices of paraffin containing the tissue,typically from 2 to 10 microns thick. The specimen slice is then appliedto a microscope slide, air dried, and heated to cause the specimen toadhere to the glass slide. Residual paraffin is then dissolved with asuitable solvent, typically xylene, toluene, or others. These so-calleddeparaffinizing solvents are then removed with a washing-dehydratingtype reagent prior to analysis of the sample by any of the techniquesdiscussed below. Alternatively, slices may be prepared from frozenspecimens, fixed briefly in 10% formalin or other suitable fixative, andthen infused with dehydrating reagent prior to analysis of the sample.

In a traditional DNA hybridization experiment using a histologicalsample, formalin-fixed paraffin embedded tissue specimens are cut intosections of 2-6 μm and collected on slides. The paraffin is melted(e.g., 30-60 minutes at 60° C.) and then removed (deparaffinated) bywashing with xylene (or a xylene substitute), e.g., 2×5 minutes. Thesamples are rehydrated, washed, and then pre-treated (e.g., 10 minutesat 95-100° C.). The slides are washed and then treated with pepsin oranother suitable permeabilizer, e.g., 3-15 minutes at 37° C. The slidesare washed (e.g., 2×3 minutes), dehydrated, and probe is applied. Thespecimens are covered with a coverslip and the slide is optionallyincubated at a temperature sufficient to denature any double-strandednucleic acid in the specimen (e.g. 5 minutes at 82° C.), followed byincubation at a temperature sufficient to allow hybridization (e.g.,overnight at 45° C.). After hybridization, the coverslips are removedand the specimens are subjected to a high-stringency wash (e.g., 10minutes at 65° C.) followed by a series of low-stringency washes (e.g.,2×3 minutes at room temperature). The samples are then dehydrated andmounted for analysis.

In a traditional RNA hybridization experiment using a histologicalsample, slides with FFPE tissue sections are deparaffinized in xylenefor 2×5 min, immerged in 99% ethanol 2×3 min, in 96% ethanol 2×3 min,and then in pure water for 3 min. Slides are placed in a humiditychamber, Proteinase K is added, and slides are incubated at RT for 5min-15 min. Slides are immersed in pure water for 2×3 min, immersed in96% ethanol for 10 sec, and air-dried for 5 min. Probes are added to thetissue section and covered with coverslip. The slides are incubated at55° C. in humidity chamber for 90 min. After incubation, the slides areimmersed in a Stringent Wash solution at 55° C. for 25 min, and thenimmersed in TBS for 10 sec. The slides are incubated in a humiditychamber with antibody for 30 min. The slides are immersed in TBS for 2×3min, then in pure water for 2×1 min, and then placed in a humiditychamber. The slides are then incubated with substrate for 60 min, andimmersed in tap water for 5 min.

In a traditional northern blot procedure, the RNA target sample isdenatured for 10 minutes at 65° C. in RNA loading buffer and immediatelyplaced on ice. The gels are loaded and electrophoresed with 1×MOPSbuffer (10×MOPS contains 200 mM morpholinopropansulfonic acid, 50 mMsodium acetate, 10 mM EDTA, pH 7.0) at 25 V overnight. The gel is thenpre-equilibrated in 20×SSC for 10 min and the RNA is transferred to anylon membrane using sterile 20×SSC as transfer buffer. The nucleicacids are then fixed on the membrane using, for example, UV-crosslinking at 120 mJ or baking for 30 min at 120° C. The membrane is thenwashed in water and air dried. The membrane is placed in a sealableplastic bag and prehybridized without probe for 30 min at 68° C. Theprobe is denatured for 5 min at 100° C. and immediately placed on ice.Hybridization buffer (prewarmed to 68° C.) is added and the probe ishybridized at 68° C. overnight. The membrane is then removed from thebag and washed twice for 5 min each with shaking in a low stringencywash buffer (e.g., 2×SSC, 0.1% SDS) at room temperature. The membrane isthen washed twice for 15 min each in prewarmed high stringency washbuffer (e.g., 0.1×SSC, 0.1% SDS) at 68° C. The membrane may then bestored or immediately developed for detection.

Additional examples of traditional hybridization techniques can befound, for example, in Sambrook et al., Molecular Cloning A LaboratoryManual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press, (1989) atsections 1.90-1.104, 2.108-2.117, 4.40-4.41, 7.37-7.57, 8.46-10.38,11.7-11.8, 11.12-11.19, 11.38, and 11.45-11.57; and in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1998)at sections 2.9.1-2.9.6, 2.10.4-2.10.5, 2.10.11-2.10.16, 4.6.5-4.6.9,4.7.2-4.7.3, 4.9.7-4.9.15, 5.9.18, 6.2-6.5, 6.3, 6.4, 6.3.3-6.4.9,5.9.12-5.9.13, 7.0.9, 8.1.3, 14.3.1-14.3.4, 14.9, 15.0.3-15.0.4,15.1.1-15.1.8, and 20.1.24-20.1.25.

(2) Hybridization Techniques

The compositions and methods of the present invention can be used fullyor partly in all types of nucleic acid hybridization techniques known inthe art for cytological and histological samples. Such techniquesinclude, for example, in situ hybridization (ISH), fluorescent in situhybridization (FISH; including multi-color FISH, Fiber-FISH, etc.),chromogenic in situ hybridization (CISH), silver in situ hybridization(SISH), comparative genome hybridization (CGH), chromosome paints, andarrays in situ.

Molecular probes that are suitable for use in the methods of theinvention may be directly or indirectly labeled with detectablecompounds such as enzymes, chromophores, fluorochromes, and haptens. DNAprobes may be present at concentrations of 0.1 to 100 ng/μL. Forexample, in some embodiments, the probes may be present atconcentrations of 1 to 10 ng/μL. PNA probes may be present atconcentrations of 0.5 to 5000 nM. For example, in some embodiments, theprobes may be present at concentrations of 5 to 1000 nM.

Molecular probes that are suitable for use in the methods of theinvention are described, e.g., in U.S. Patent Publication No.2005/0266459, which is incorporated herein by reference. In general,probes may be prepared by chemical synthesis, PCR, or by amplifying aspecific DNA sequence by cloning, inserting the DNA into a vector, andamplifying the vector an insert in appropriate host cells. Commonly usedvectors include bacterial plasmids, cosmids, bacterial artificialchromosomes (BACs), PI diverted artificial chromosomes (PACs), or yeastartificial chromosomes (YACs). The amplified DNA is then extracted andpurified for use as a probe. Methods for preparing and/or synthesizingprobes are known in the art, e.g., as disclosed in PCT/US02/30573.

The nucleic acid probe may be a double or single stranded nucleic acidfragment or sequence, such as a DNA, RNA, or analogs such as PNA or LNA.The probes may be labeled to make identification of the probe-targethybrid possible by use, for example, of a fluorescence or bright fieldmicroscope/scanner. In some embodiments, the probe may be labeled usingradioactive labels such as ³¹P, ³³P, or ³²S, non-radioactive labels suchas digoxigenin and biotin, or fluorescent labels

In general, the type of probe determines the type of feature one maydetect in a hybridization assay. For example, total nuclear or genomicDNA probes can be used as a species-specific probe. Chromosome paintsare collections of DNA sequences derived from a single chromosome typeand can identify that specific chromosome type in metaphase andinterphase nuclei, count the number of a certain chromosome, showtranslocations, or identify extra-chromosomal fragments of chromatin.Different chromosomal types also have unique repeated sequences that maybe targeted for probe hybridization, to detect and count specificchromosomes. Large insert probes may be used to target uniquesingle-copy sequences. With these large probes, the hybridizationefficiency is inversely proportional to the probe size. Smaller probescan also be used to detect aberrations such as deletions,amplifications, inversions, duplications, and aneuploidy. For example,differently-colored locus-specific probes can be used to detecttranslocations via split-signal in situ hybridization.

In general, the ability to discriminate between closely relatedsequences is inversely proportional to the length of the hybridizationprobe because the difference in thermal stability decreases between wildtype and mutant complexes as probe length increases. Probes of greaterthan 10 bp in length are generally required to obtain the sequencediversity necessary to correctly identify a unique organism or clinicalcondition of interest. On the other hand, sequence differences as subtleas a single base (point mutation) in very short oligomers (<10 basepairs) can be sufficient to enable the discrimination of thehybridization to complementary nucleic acid target sequences as comparedwith non-target sequences.

In one embodiment, at least one set of the in situ hybridization probesmay comprise one or more PNA probes, as defined above and as describedin U.S. Pat. No. 7,105,294, which is incorporated herein by reference.Methods for synthesizing PNA probes are described in PCT/US02/30573.Alternatively, or in addition, at least one set of the hybridizationprobes in any of the techniques discussed above may comprise one or morelocked nucleic acid (LNA) probes, as described in WO 99/14226, which isincorporated herein by reference. Due to the additional bridging bondbetween the 2′ and 4′ carbons, the LNA backbone is pre-organized forhybridization. LNA/DNA and LNA/RNA interactions are stronger than thecorresponding DNA/DNA and DNA/RNA interactions, as indicated by a highermelting temperature. Thus, the compositions and methods of theinvention, which decrease the energy required for hybridization, areparticularly useful for hybridizations with LNA probes.

In one embodiment, the probes may comprise a detectable label (amolecule that provides an analytically identifiable signal that allowsthe detection of the probe-target hybrid), as described in U.S. PatentPublication No. 2005/0266459, which is incorporated herein by reference.The detectable label may be directly attached to a probe, or indirectlyattached to a probe, e.g., by using a linker. Any labeling method knownto those in the art, including enzymatic and chemical processes, can beused for labeling probes used in the methods and compositions of theinvention. In other embodiments, the probes are not labeled.

In general, in situ hybridization techniques such as FISH, CISH, andSISH for DNA detection, employ large, mainly unspecified, nucleic acidprobes that hybridize with varying stringency. Using large probesrenders the in situ hybridization technique very sensitive. However, thesuccessful use of large probes in traditional hybridization assaysdepends on blocking the undesired background staining derived from,e.g., repetitive sequences that are present throughout the genome.Traditional methods for decreasing nonspecific probe binding includesaturating the binding sites on proteins and tissue by incubating tissuewith prehybridization solutions containing ficoll, bovine serum albumin(BSA), polyvinyl pyrrolidone, and nucleic acids. Such blocking steps aretime-consuming and expensive. As discussed below, the methods andcompositions of the invention advantageously reduce and/or eliminate theneed for such blocking steps and blocking reagents. However, in oneembodiment, repetitive sequences may be suppressed according to themethods known in the art, e.g., as disclosed in PCT/US02/30573.

Bound probes may be detected in cytological and histological sampleseither directly or indirectly with fluorochromes (e.g., FISH), organicchromogens (e.g., CISH), silver particles (e.g., SISH), or othermetallic particles (e.g., gold-facilitated fluorescence in situhybridization, GOLDFISH). Thus, depending on the method of detection,populations of cells obtained from a sample to be tested may bevisualized via fluorescence microscopy or conventional brightfield lightmicroscopy.

Hybridization assays on cytological and histological samples areimportant tools for determining the number, size, and/or location ofspecific DNA sequences. For example, in CGH, whole genomes are stainedand compared to normal reference genomes for the detection of regionswith aberrant copy number. Typically, DNA from subject tissue and fromnormal control tissue is labeled with different colored probes. Thepools of DNA are mixed and added to a metaphase spread of normalchromosomes (or to a microarray chip, for array- or matrix-CGH). Theratios of colors are then compared to identify regions with aberrantcopy number.

FISH is typically used when multiple color imaging is required and/orwhen the protocol calls for quantification of signals. The techniquegenerally entails preparing a cytological sample, labeling probes,denaturing target chromosomes and the probe, hybridizing the probe tothe target sequence, and detecting the signal. Typically, thehybridization reaction fluorescently stains the targeted sequences sothat their location, size, or number can be determined usingfluorescence microscopy, flow cytometry, or other suitableinstrumentation. DNA sequences ranging from whole genomes down toseveral kilobases can be studied using FISH. With enhanced fluorescencemicroscope techniques, such as, for example, deconvolution, even asingle mRNA molecule can be detected. FISH may also be used on metaphasespreads and interphase nuclei.

FISH has been used successfully for mapping repetitive and single-copyDNA sequences on metaphase chromosomes, interphase nuclei, chromatinfibers, and naked DNA molecules, and for chromosome identification andkaryotype analysis through the localization of large repeated families,typically the ribosomal DNAs and major tandem array families. One of themost important applications for FISH has been in detecting single-copyDNA sequences, in particular disease related genes in humans and othereukaryotic model species, and the detection of infections agents. FISHmay be used to detect, e.g., chromosomal aneuploidy in prenataldiagnoses, hematological cancers, and solid tumors; gene abnormalitiessuch as oncogene amplifications, gene deletions, or gene fusions;chromosomal structural abnormalities such as translocations,duplications, insertions, or inversions; contiguous gene syndromes suchas microdeletion syndrome; the genetic effects of various therapies;viral nucleic acids in somatic cells and viral integration sites inchromosomes; etc. In multi-color FISH, each chromosome is stained with aseparate color, enabling one to determine the normal chromosomes fromwhich abnormal chromosomes are derived. Such techniques includemultiplex FISH (m-FISH), spectral karyotyping (SKY), combined binaryration labeling (COBRA), color-changing karyotyping, cross-species colorbanding, high resolution multicolor banding, telomeric multiplex FISH(TM-FISH), split-signal FISH (ssFISH), and fusion-signal FISH.

CISH and SISH may be used for many of the same applications as FISH, andhave the additional advantage of allowing for analysis of the underlyingtissue morphology, for example in histopathology applications. If FISHis performed, the hybridization mixture may contain sets of distinct andbalanced pairs of probes, as described in U.S. Pat. No. 6,730,474, whichis incorporated herein by reference. For CISH, the hybridization mixturemay contain at least one set of probes configured for detection with oneor more conventional organic chromogens, and for SISH, the hybridizationmixture may contain at least one set of probes configured for detectionwith silver particles, as described in Powell R D et al.,“Metallographic in situ hybridization,” Hum. Pathol., 38:1145-59 (2007).

The compositions of the invention may also be used fully or partly inall types of molecular biology techniques involving hybridization,including blotting and probing (e.g., Southern, northern, etc.), andarrays.

(3) Conditions for Performing Hybridization Assays

The method of the present invention involves the use of polar aproticsolvents in a stringent wash buffer for hybridization applications. Thecompositions of the present invention are particularly useful in saidmethod.

A great variation exists in the traditional hybridization protocolsknown in the art. The stringent wash compositions of the invention maybe used in any of the traditional hybridization protocols known in theart. For example, the heat pre-treatment, digestion, denaturation,hybridization, washes, and mounting steps may use the same conditions interms of volumes, temperatures, reagents (aside from the stringent washbuffer), and incubation times as for traditional hybridizationapplications.

For example, in some embodiments, the denaturation temperature may varyfrom 60 to 100° C. and the hybridization temperature may vary from 20 to60° C. In other embodiments, the denaturation temperature may vary from60 to 70° C., 70 to 80° C., 80 to 90° C. or 90 to 100° C., and thehybridization temperature may vary from 20 to 30° C., 30 to 40° C., 40to 50° C., or 50 to 60° C. In other embodiments, the denaturationtemperature is 72, 82, or 92° C., and the hybridization temperature is37, 40, 45, or 50° C.

In other embodiments, the denaturation time may vary from 0 to 10minutes and the hybridization time may vary from 0 minutes to 24 hours.In other embodiments, the denaturation time may be from 0 to 5 minutesand the hybridization time may be from 0 minute to 8 hours. In otherembodiments, the denaturation time is 0, 1, 2, 3, 4, or 5 minutes, andthe hybridization time is 0 minutes, 5 minutes, 15 minutes, 30 minutes,60 minutes, 180 minutes, or 240 minutes. It will be understood by thoseskilled in the art that in some cases, e.g., RNA detection, adenaturation step is not required.

Alternatively, assays using the compositions of the invention can bechanged and optimized from traditional methodologies, for example, bydecreasing the stringent wash time and/or decreasing the stringent washtemperatures.

After complementary strands of nucleic acid have reannealed in ahybridization application, the hybridization product will generallycomprise complementary base pairing and non-complementary base pairingbetween the probe and the target nucleic acid. Any non-complementarybase pairing is then removed by a series of post-hybridization washes.Four variables are typically adjusted to influence the stringency of thepost-hybridization washes:

-   -   1. Temperature (as temperature increases, non-perfect matches        between the probe and the target sequence will denature, i.e.,        separate, before more perfectly matched sequences).    -   2. Salt conditions (as salt concentration decreases, non-perfect        matches between the probe and the target sequence will denature,        i.e., separate, before more perfectly matched sequences).    -   3. Formamide concentration (as the amount of formamide        increases, non-perfect matches between the probe and the target        sequence will denature, i.e., separate, before more perfectly        matched sequences).    -   4. Time (as the wash time increases, non-perfect matches between        the probe and the target sequence will denature, i.e., separate,        before more perfectly matched sequences).

Stringent wash methods using the compositions of the invention mayinvolve applying the compositions to a hybridization product comprisinga target nucleic acid sequence hybridized to a probe. During thestringent wash step, the polar aprotic solvent interacts with thehybridization product and facilitates the denaturation of the mismatched(i.e., non-complementary) sequences. The polar aprotic solventsspecified in the present invention speed up this process, reduce thetemperature required for the stringency wash, and reduce the harshnessand toxicity of the stringency wash conditions compared toformamide-containing buffers.

Those skilled in the art will understand that different type ofhybridization assays, different types of samples, different types ofprobe targets, different length of probes, different types of probes,e.g. DNA,/RNA/PNA/LNA oligos, short DNA/RNA probes (0.5-3 kb),chromosome paint probes, CGH, repetitive probes (e.g. alpha-satelliterepeats), single-locus etc., will effect the concentrations of e.g. saltand polar aprotic solvents required to obtain the most effectivepost-hybridization washes. The temperature and incubation time are alsoimportant variables for stringent washes using the compositions of theinvention. In view of the guidance provided herein, one skilled in theart will understand how to vary these factors to optimize the stringencywashes in hybridization applications.

The concentration of probe may be varied in order to produce strongsignals and/or reduce background. For example, reducing probeconcentration reduces background. However, reducing the probeconcentration is inversely related to the hybridization time, i.e., thelower the concentration, the higher hybridization time required.Background levels can also be reduced by adding agents that reducenon-specific binding, such as to the cell membrane, such as smallamounts of total human DNA or non-human-origin DNA (e.g., salmon spermDNA) to a hybridization reaction using the compositions of theinvention. However, the compositions of the invention often allow forbetter signal-to-noise ratios than traditional stringent washcompositions.

Traditional assay methods may also be changed and optimized when usingthe compositions of the invention depending on whether the system ismanual, semi-automated, or fully automated. For example, asemi-automated or fully automated system will benefit from the lowertemperature and shorter stringency washes obtained with the compositionsof the invention. These features may reduce the difficulties encounteredwhen traditional compositions are used in such systems. For example, oneproblem with semi-automated and fully automated systems is thatsignificant evaporation of the sample can occur during the stringentwash step, since such systems require small sample volumes (e.g., 150μL) and elevated temperatures. Thus, proportions of the components intraditional hybridization compositions are fairly invariable. However,since the compositions of the invention allow for stringent washes atlower temperatures, evaporation is reduced, allowing for increasedflexibility in the proportions of the components in hybridizationcompositions used in semi-automated and fully automated systems.

Thus, the compositions and methods of the invention solve many of theproblems associated with traditional hybridization compositions andmethods.

The disclosure may be understood more clearly with the aid of thenon-limiting examples that follow, which constitute preferredembodiments of the compositions according to the disclosure. Other thanin the examples, or where otherwise indicated, all numbers expressingquantities of ingredients, reaction conditions, and so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained herein. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope are approximations, the numerical values set forth inthe specific example are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in its respective testingmeasurements. The examples that follow illustrate the present inventionand should not in any way be considered as limiting the invention.

EXAMPLES

Reference will now be made in detail to specific embodiments of theinvention. While the invention will be described in conjunction withthese embodiments, it will be understood that they are not intended tolimit the invention to those embodiments. On the contrary, the inventionis intended to cover alternatives, modifications, and equivalents, whichmay be included within the invention as defined by the appended claims.

The reagents used in the following examples are from Dako's HistologyFISH Accessory Kit (K5599) and Cytology FISH Accessory Kit (K5499) (DakoDenmark A/S, Glostrup Denmark). The kits contain all the key reagents,except for probe, required to complete a FISH procedure forformalin-fixed, paraffin-embedded tissue section specimens. All sampleswere prepared according to the manufacturer's description. The DakoHybridizer (S2451, Dako) was used for the digestion, denaturation, andhybridization steps.

Evaluation of FISH slides was performed within a week afterhybridization using a Leica DM6000B fluorescence microscope, equippedwith DAPI, FITC, Texas Red single filters and FITC/Texas Red doublefilter under 10×, 20×, 40×, and 100× oil objective.

Evaluation of CISH slides was performed using an Olympus BX51 lightmicroscope, under 4×, 10×, 20×, 40×, and 60× objective.

In the Examples that follow, “dextran sulfate” refers to the sodium saltof dextran sulfate (D8906, Sigma) having a molecular weightM_(w)>500,000. All concentrations of polar aprotic solvents are providedas v/v percentages. Phosphate buffer refers to a phosphate bufferedsolution containing NaH₂PO₄, 2H₂O (sodium phosphate dibasic dihydrate)and Na₂HPO₄, H₂O (sodium phosphate monobasic monohydrate). Citratebuffer refers to a citrate buffered solution containing sodium citrate(Na₃C₆H₅O₇, 2H₂O; 1.06448, Merck) and citric acid monohydrate (C₆H₈O₇,H₂O; 1.00244, Merck).

General Histology FISH/CISH Procedure for Examples 1-20

The slides with cut formalin-fixed paraffin embedded (FFPE) multipletissue array sections from humans (tonsils, mammacarcinoma, kidney andcolon) were baked at 60° C. for 30-60 min, deparaffinated in xylenebaths, rehydrated in ethanol baths and then transferred to Wash Buffer.The samples were then pre-treated in Pre-Treatment Solution at a minimumof 95° C. for 10 min and washed 2×3 min. The samples were then digestedwith Pepsin RTU at 37° C. for 3 min, washed 2×3 min, dehydrated in aseries of ethanol evaporations, and air-dried. The samples were thenincubated with 10 μL FISH probe as described under the individualexperiments. The samples were then washed by Stringency Wash at 65° C.10 min, then washed 2×3 min, then dehydrated in a series of ethanolevaporations, and air-dried. Finally, the slides were mounted with 15 μLAntifade Mounting Medium. When the staining was completed, observerstrained to assess signal intensity, morphology, and background of thestained slides performed the scoring.

General Cytology FISH Procedure for Examples 21 and 22

Slides with metaphases preparation were fixed in 3.7% formaldehyde for 2min, washed 2×5 min, dehydrated in a series of ethanol evaporations, andair-dried. The samples were then incubated with 10 μL FISH probe asdescribed under the individual experiments. The samples were then washedby Stringency Wash at 65° C. 10 min, then washed 2×3 min, thendehydrated in a series of ethanol evaporations, and air-dried. Finally,the slides were mounted with 15 μL Antifade Mounting Medium. When thestaining was completed, observers trained to assess signal intensity andbackground of the stained slides performed the scoring as described inthe scoring for guidelines for tissue sections.

General Histology FISH/CISH Procedure for Examples 23-26

Slides with cut formalin-fixed paraffin embedded (FFPE) multiple tissuearray sections from humans (tonsils, mammacarcinoma, kidney and colon)were baked at 60° C. for 30-60 min, deparaffinated in xylene baths,rehydrated in ethanol baths and then transferred to Wash Buffer. Thesamples were then pre-treated in Pre-Treatment Solution at a minimum of95° C. for 10 min and washed 2×3 min. The samples were then digestedwith Pepsin RTU at 37° C. for 3 min, washed 2×3 min, dehydrated in aseries of ethanol evaporations, and air-dried. The samples were thenincubated with 10 μL FISH probe denatured at 82° C. for 5 min andhybridized at 45° C. as described under the individual experiments. Thesamples were then washed as described under the individual experiments,dehydrated in a series of ethanol evaporations, and air-dried. Finally,the slides were mounted with 15 μL Antifade Mounting Medium. When thestaining was completed, observers trained to assess signal intensity,morphology, and background of the stained slides performed the scoring.

Scoring Guidelines of Tissue Sections

The signal intensities were evaluated on a 0-3 scale with 0 meaning nosignal and 3 equating to a strong signal. The cell/tissue structures areevaluated on a 0-3 scale with 0 meaning no structure and no nucleiboundaries and 3 equating to intact structure and clear nucleiboundaries. Between 0 and 3 there are additional grades 0.5 apart fromwhich the observer can assess signal intensity, tissue structure, andbackground.

The signal intensity is scored after a graded system on a 0-3 scale.

0 No signal is seen. 1 The signal intensity is weak. 2 The signalintensity is moderate. 3 The signal intensity is strong.

The scoring system allows the use of ½ grades.

The tissue and nuclear structure is scored after a graded system on a0-3 scale.

0 The tissue structures and nuclear borders are completely destroyed. 1The tissue structures and/or nuclear borders are poor. This gradeincludes situations where some areas have empty nuclei. 2 Tissuestructures and/or nuclear borders are seen, but the nuclear borders areunclear. This grade includes situations where a few nuclei are empty. 3Tissue structures and nuclear borders are intact and clear.

The scoring system allows the use of ½ grades.

The background is scored after a graded system on a 0-3 scale.

0 Little to no background is seen. 1 Some background. 2 Moderatebackground. 3 High Background.

The scoring system allows the use of ½ grades.

Example 1

This example compares the signal intensity and cell morphology fromsamples treated with the compositions of the invention or traditionalhybridization solutions as a function of denaturation temperature.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide (15515-026, Invitrogen), 5 μM blockingPNAs (see Kirsten Vang Nielsen et al., PNA Suppression Method Combinedwith Fluorescence In Situ Hybridisation (FISH) Technique in PRINS andPNA Technologies in Chromosomal Investigation, Chapter 10 (FranckPellestor ed.) (Nova Science Publishers, Inc. 2006)), 10 ng/μL Texas Redlabeled CCND1 gene DNA probe (RP11-1143E20, size 192 kb).

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate (03519, Fluka), 5 μM blockingPNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe (RP11-1143E20,size 192 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were denatured as indicated for 5 min and hybridized at 45°C. for 60 minutes.

Results:

Signal Denaturation (I) (II) Cell morphology temperature Formamide ECFormamide EC 72° C. 0 2 Good Good 82° C. ½ 3 Good Good 92° C. ½ 3 Notgood Not good Signals scored as “3” were clearly visible in a 20xobjective.

Example 2

This example compares the signal intensity and background staining fromsamples treated with the compositions of the invention or traditionalhybridization solutions as a function of hybridization time.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Redlabeled CCND1 gene DNA probe.

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, 5 μM blocking PNAs, 10 ng/μLTexas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 14hours, 4 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 0 minutes.

Results:

Signal Hybridization (I) (II) Background staining time Formamide ECFormamide EC 14 hours 3 3 +½ +2  4 hours 1 3 +½ +1  2 hours ½ 3 +0 +1 60min. ½ 3 +0 +1 30 min. 0 2½ +0 +1 15 min. 0 2 +0 +1  0 min. 0 1 +0 +½Signals scored as “3” were clearly visible in a 20x objective.

Example 3

This example compares the signal intensity from samples treated with thecompositions of the invention having different polar aprotic solvents ortraditional hybridization solutions.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Redlabeled CCND1 gene DNA probe.

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate (EC), 5 μM blocking PNAs, 10ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition III: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Propylene carbonate (PC) (540013, Aldrich), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition IV: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Sulfolane (SL) (T22209, Aldrich), 5 μM blockingPNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition V: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Aceto nitrile (AN) (C02CIIX, Lab-Scan), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition VI: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% γ-butyrolactone (GBL) (B103608, Aldrich), 5 μMblocking PNAs, 7,5 ng/μL Texas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Signal (I) (II) (III) (IV) (V) (VI) Formamide EC PC SL AN GBL ½ 3 3 3 23 Signals scored as “3” were clearly visible in a 20x objective.

Example 4

This example compares the signal intensity from samples treated with thecompositions of the invention having different concentrations of polaraprotic solvent.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 10-60% Ethylene carbonate (as indicated), 5 μMblocking PNAs, 7.5 ng/μL Texas Red labeled IGK-constant DNA gene probe((CTD-3050E15, RP11-1083E8; size 227 kb) and 7.5 ng/μL FITC labeledIGK-variable gene DNA probe (CTD-2575M21, RP11-122B6, RP11-316G9; size350 and 429 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Ethylene carbonate (EC) 10% 20% 30% 40% 60% Signal Texas Red 1½ 2 3 3 2intensity FITC 1 1½ 2 2½ 2 Signals scored as “3” were clearly visible ina 20x objective.

Example 5

This example compares the signal intensity and background intensity fromsamples treated with the compositions with and without PNA blocking.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, 7.5 ng/μL Texas Red labeledCCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Ethylene carbonate (EC) PNA- blocking Non- PNA blocking Signal intensity3 3 Background intensity ½+ ½+ Signals scored as “3” were clearlyvisible in a 20x objective.

Example 6

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of probe concentration andhybridization time.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, and 10, 7.5, 5 or 2.5 ng/μLTexas Red labeled CCND1 gene DNA probe (as indicated).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 3hours, 2 hours and 1 hours.

Results:

Signal Intensity Hybridization (I) (II) (III) (IV) time 10 ng/μL 7.5ng/μL 5 ng/μL 2.5 ng/μL 3 hours 3 3 3 3 2 hours 3 3 3 1 1 hours 3 3 3 ½Signals scored as “3” were clearly visible in a 20x objective.

Example 7

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of salt, phosphate, andbuffer concentrations.

FISH Probe Compositions: 10% dextran sulfate, ([NaCl], [phosphatebuffer], [TRIS buffer] as indicated in Results), 40% Ethylene carbonate,7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

[NaCl] 300 mM 100 mM 0 mM Signal intensity 2 1 ½ phosphate [0 mM] Signalintensity 3 2½ ½ phosphate [5 mM] Signal intensity — — 3 phosphate [35mM] Signal intensity — — 2 TRIS [40 mM] Signals scored as “3” wereclearly visible in a 20x objective.

Example 8

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of dextran sulfateconcentration.

FISH Probe Compositions: 0, 1, 2, 5, or 10% dextran sulfate (asindicated), 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate,5 ng/μL Texas Red labeled SIL-TAL1 gene DNA probe (RP1-278O13; size 67kb) and 6 ng/μL FITC SIL-TAL1 (ICRFc112-112C1794, RP11-184J23, RP11-8J9,CTD-2007B18, 133B9; size 560 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes. No blocking.

Results:

Signal Intensity % Dextran Sulfate Texas Red Probe FITC Probe 0% 1 1 1%1 1 2% 1½ 1½ 5% 2 2½ 10%  2 2½ NOTE: this experiment did not produceresults scored as “3” because the SIL-TAL1 Texas Red labeled probe isonly 67 kb and was from a non-optimized preparation.

Example 9

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of dextran sulfate, salt,phosphate, and polar aprotic solvent concentrations.

FISH Probe Composition Ia: 34% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ib: 34% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ic: 34% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIa: 32% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIb: 32% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIc: 32% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ma: 30% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IIIb: 30% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IIIc: 30% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVa: 28% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVb: 28% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVc: 28% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Reference V: Standard sales vial of HER2 PharmDx probe mix(K5331, Dako) containing blocking PNA. Overnight hybridization for 20hours.

All compositions were present as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes with noblocking, except for FISH Probe Reference V, which had PNA blocking andwas hybridized for 20 hours.

Results:

Signal Strength DNA Probes PNA Probes Composition Ia 0  ½ Composition Ib0  ½ Composition Ic ½ 2½ Composition IIa ½ 3 Composition IIb 1 2Composition IIc ½ 3 Composition IIIa 1 2½ Composition IIIb 1½  2½Composition IIIc 2 3 Composition IVa 2½-3 3 Composition IVb 3 3Composition IVc 3 3 Reference V 2 2½ NOTE: Composition IVa gave strongDNA signals with no salt. This is not possible with standard FISHcompositions, where DNA binding is salt dependent.

Example 10

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of polar aprotic solvent anddextran sulfate concentration under high salt (4× normal) conditions.

FISH Probe Composition I: 0% ethylene carbonate, 29% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition II: 5% ethylene carbonate, 27% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition III: 10% ethylene carbonate, 25% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition IV (not tested): 20% ethylene carbonate, 21%dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL TexasRed labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNAprobe. Composition had two phases.

Results:

Signal Strength DNA Probes PNA Probes Composition I ½ 3 Composition II 22½ Composition III 3 3 Composition IV — — Note: Composition II gave goodDNA signals with only 5% EC and strong DNA signals with 10% EC.

Example 11

This example compares the signal intensity and background from samplestreated with different phases of the compositions of the invention.

FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphatebuffer, 40% Ethylene carbonate, 8 ng/μL Texas Red labeled HER2 gene DNAprobe and 600 nM FITC-labeled CEN-17 PNA probe. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes. Noblocking.

Results:

Signal Intensity DNA Probe PNA Probe Background Upper Phase 3 1½ +2Lower Phase 3 2½ +1 Mix of Upper and 2½ 3 +½ Lower Phases NOTE: theupper phase had more background than the lower phase in theseexperiments.

Example 12

This example is similar to the previous example, but uses a differentDNA probe and GBL instead of EC.

FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphatebuffer, 40% GBL, 10 ng/μL Texas Red labeled CCND1 gene DNA probe and 600nM FITC-labeled CEN-17 PNA probe.

The FISH probes were incubated at 82° C. for 5 min and then at 45° C.for 60 minutes. No blocking.

Results:

Signal Strength DNA Probe PNA Probe Background Top Phase 3 0-½ +1½Bottom Phase 2 ½ +3 Mixed Phases 2½ ½ +2½

Example 13

This example examines the number of phases in the compositions of theinvention as a function of polar aprotic solvent and dextran sulfateconcentration.

FISH Probe Compositions: 10 or 20% dextran sulfate; 300 mM NaCl; 5 mMphosphate buffer; 0, 5, 10, 15, 20, 25, 30% EC; 10 ng/μL probe.

Results:

Number of Phases Number of Phases % EC 10% Dextran 20% Dextran 0 1 1 5 11 10 1 1 15 1 1 20 2 2 25 2 2 30 2 2 NOTE: 15% EC, 20% dextran sulfateproduces the nicest high signal intensities of the above one phasesolution. Two phases 20% EC has even higher signal intensities than 15%.(Data not shown).

Example 14

This example compares the signal intensity and background from samplestreated with different compositions of the invention as a function ofprobe concentration and hybridization time.

FISH Probe Composition I: 10 ng/μL HER2 TxRed labeled DNA probe(standard concentration) and standard concentration of CEN7 FITC labeledPNA probe (50 nM); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

FISH Probe Composition II: 5 ng/μL HER2 TxRed labeled DNA probe (½ ofstandard concentration) and standard concentration (50 nM) of FITClabeled CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

FISH Probe Composition III: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (25 nM) ofCEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

Compositions I-III existed as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 3 hours, 2 hoursand 1 hours.

Results:

Hybrid- Signal Intensity ization I II III time DNA PNA B.G. DNA PNA E.G.DNA PNA B.G. 3 hours 3 3 +3 3 3 +2.5 3 3 +1.5 2 hours 2.5 2.5 +3 3 3 +33 3 +1.5 1 hours 2.5 2.5 +3 3 3 +1.5 2.5 3 +1 Signals scored as “3” wereclearly visible in a 20x objective. B.G.: Back ground.

Example 15

This example compares the signal intensity and background from samplestreated with the compositions of the invention as a function of blockingagent.

FISH Probe Compositions: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer; 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ of standardconcentration) and ½ of the standard concentration (300 nM) FITC labeledCEN17 PNA probe. Samples were blocked with: (a) nothing; (b) 0.1 μg/μLCOT1 (15279-011, Invitrogen); (c) 0.3 μg/μL COT1; or (d) 0.1 μg/μL totalhuman DNA before hybridization using the compositions of the invention.

All samples were present as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes.

Results:

Signal Intensity Blocking Agent Background DNA PNA Nothing +1-1.5 3 2.50.1 μg/μL COT1 +1 3 2.5 0.3 μg/μL COT1 +1.5 3 2.5 0.1 μg/μL total humanDNA +½ 3 2.5 NOTE: Background levels without blocking are significantlylower than what is normally observed by standard FISH with no blocking.In contrast, if a standard FISH composition does not contain a blockingagent, signals normally cannot be read.

Example 16

This experiment compares different ways of removing background stainingusing the compositions of the invention.

All compositions contained 15% EC, 20% dextran sulfate, 600 mM NaCl, 10mM phosphate buffer, 2.5 ng/μL HER2 DNA probes (¼ of standardconcentration), 300 nM CEN17 PNA probe (½ of standard concentration),and one of the following background-reducing agents:

A) 5 μM blocking-PNA (see Kirsten Vang Nielsen et al., PNA SuppressionMethod Combined with Fluorescence In Situ Hybridisation (FISH) Techniquein PRINS and PNA Technologies in Chromosomal Investigation, Chapter 10(Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006))B) 0.1 μg/μL COT-1 DNAC) 0.1 μg/μL total human DNA (THD) (sonicated unlabelled THD)D) 0.1 μg/μL sheared salmon sperm DNA (AM9680, Ambion)E) 0.1 μg/μL calf thymus DNA (D8661, Sigma)F) 0.1 μg/μL herring sperm DNA (D7290, Sigma)G) 0.5% formamideH) 2% formamideI) 1% ethylene glycol (1.09621, Merck)J) 1% glycerol (1.04095, Merck)

K) 1% 1,3-Propanediol (533734, Aldrich)

L) 1% H₂0 (control)

All samples were present as a single phase. The probes were incubated at82° C. for 5 minutes and then at 45° C. on FFPE tissue sections for 60and 120 minutes.

Results:

Signal Intensity Background blocking Hybridization/min Background DNAPNA Blocking-PNA 60 +1 3 2.5 Blocking-PNA 120 +1-1½ 3 2.5 COT-1 60 +½ 32.5 COT-1 120 +0-½  3 2.5 THD 60 +0 3 3 THD 120 +½ 3 2.5 Salmon DNAsperm 60 +0 3 3 Salmon DNA sperm 120 +0 3 3 Calf Thymus DNA 60 +0 2.5 3Calf Thymus DNA 120 +½ 3 2.5 Hearing sperm DNA 60 +0 3 3 Hearing spermDNA 120 +½ 2.5 3 0.5% formamide 60 +0 2.5 3 0.5% formamide 120 +0 3 3 2%formamide 60 +½ 2.5 3 2% formamide 120 +½ 3 3 1% Ethylene Glycol 60 +½2.5 3 1% Ethylene Glycol 120 +1½  3 2.5 1% Glycerol 60 +½ 0.5 3 1%Glycerol 120 +1 3 2.5 1% 1,3-Propanediol 60 +0 3 2.5 1% 1,3-Propanediol120 +1 3 2.5 Nothing 60 +1 2.5 2.5 Nothing 120 +1½  3 2.5 NOTE: allbackground reducing reagents, except for blocking-PNA, showed an effectin background reduction. Thus, specific blocking against repetitive DNAsequences is not required.

Example 17

This experiment compares the signal intensity from the upper and lowerphases using two different polar aprotic solvents.

FISH Probe Composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% ethylene trithiocarbonate (ET) (E27750, Aldrich),5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe Composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% glycol sulfite (GS) (G7208, Aldrich), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

The FISH probes were incubated at 82° C. for 5 min and then at 45° C.for 60 minutes.

Results:

Signal Intensity I (ET) II (GS) Upper Phase 1½ 0 Lower Phase 0 3 Mix ofUpper and Lower Phases 2½ 3

Example 18

This experiment examines the ability of various polar aprotic solventsto form a one-phase system.

All compositions contained: 20% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, and either 10, 15, 20, or 25% of one of the followingpolar aprotic solvents:

Sulfolane γ-Butyrolactone

Ethylene trithiocarbonateGlycol sulfitePropylene carbonate

Results: all of the polar aprotic solvents at all of the concentrationsexamined produced at least a two-phase system in the compositions used.However, this does not exclude that these compounds can produce aone-phase system under other composition conditions.

Example 19

This experiment examines the use of the compositions of the invention inchromogenic in situ hybridization (CISH) analysis on multi FFPE tissuesections.

FISH Probe Composition I: 4.5 ng/μL TCRAD FITC labelled gene DNA probe(¼ of standard concentration) (RP11-654A2, RP11-246A2, CTP-2355L21,RP11-158G6, RP11-780M2, RP11-481C14; size 1018 kb); 15% EC; 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

FISH Probe Composition II: 4.5 ng/μL TCRAD FITC labelled gene DNA probe(¼ of standard concentration) (size 1018 kb); 15% EC; 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL shearedsalmon DNA sperm.

FISH Probe Composition III: 300 nM of each individual FITC labelled PNACEN17 probe (½ of standard concentration); 15% EC; 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.0.

All samples were analyzed using the Dako DuoCISH protocol (SK108) andcompositions for split probes with the exception that the stringencywash was conducted for 20 minutes instead of 10 minutes, and withoutusing the DuoCISH red chromogen step.

Results:

Signal Strength Composition FITC DNA FITC PNA I 3 — II 3 — III — 3 Note:The signal intensities were very strong. Due to the high levels ofbackground, it was not possible to discriminate if addition of salmonsperm DNA in Composition II reduced the background. Signals were clearlyvisible using a 10x objective in e.g. tonsils, which in general had lessbackground. If tissues possessed high background, the signals wereclearly visible using a 20x objective.

Example 20

This example compares the signal intensity and background from FFPEtissue sections treated with the compositions of the invention with twoDNA probes.

FISH Probe Composition I: 9 ng/μL IGH FITC labelled gene DNA probe(RP11-151B17, RP11-112H5, RP11-101G24, RP11-12F16, RP11-47P23,CTP-3087C18; size 612 kb); 6.4 ng/μL MYC Tx Red labeled DNA probe(CTD-2106F24, CTD-2151C21, CTD-2267H22; size 418 kb); 15% EC; 20%dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

FISH Probe Composition II: 9 ng/μL IGH FITC labelled gene DNA probe; 6.4ng MYC TxRed labeled DNA probe; 15% EC, 20% dextran sulfate; 600 mMNaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon sperm DNA.

Signal Strength Salmon DNA FITC probe Texas Red probe Background − 2½ 2½+2.5 + 3 3 +1.5 NOTE: the high background was probably due to the factthat standard probe concentrations were used.

Example 21

This experiment examines the use of the compositions of the invention oncytological samples.

FISH Probe Composition: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer; 5 ng/μL HER2 TxRed labeled DNA probe (½ of standardconcentration) and ½ of the standard concentration of CEN7 (25 nM).

The FISH probes were incubated on metaphase chromosome spreads at 82° C.for 5 minutes, then at 45° C. for 30 minutes, all without blocking.

Results:

Signal Strength DNA Probe PNA Probe Background 3 3 +1

No chromosome banding (R-banding pattern) was observed with thecompositions of the invention, in contrast with traditional ISHsolutions, which typically show R-banding. A low homogenously redbackground staining of the interphase nuclei and metaphase chromosomeswas observed.

Example 22

This example compares the signal intensity and background from DNAprobes on cytology samples, metaphase spreads, with and withoutblocking.

FISH Probe Composition I: 6 ng/μL TCRAD Texas Red labelled gene DNAprobe (standard concentration) (CTP-31666K20, CTP-2373N7; size 301 kb)and 4.5 ng/μL FITC labelled gene DNA probe (¼ of standardconcentration); 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citratebuffer, pH 6.0.

FISH Probe Composition II: 6 ng/μL TCRAD Texas Red labelled gene DNAprobe (standard concentration) (size 301 kb) and 4.5 ng/μL FITC labelledgene DNA probe (¼ of standard concentration); 15% EC, 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL shearedsalmon sperm DNA.

The FISH probes were incubated on metaphase spreads at 82° C. for 5 min,then at 45° C. for 60 min.

Results:

Signal Intensity Blocking Agent Background Tx Red FITC Nothing +0 3 30.1 μg/μL Salmon DNA +0 3 3

Again, no chromosome banding (R-banding pattern) was observed with thecompositions of the invention. In addition, no background staining ofthe interphase nuclei or the metaphase chromosomes were observed.

Example 23

This example compares the background and signal intensity as a functionof the stringency wash conditions.

Stringency wash composition I: 2×SSC (pH 7.0)

Stringency wash composition II: 2×SSC (pH 7.0), 50% EC (E2, 625-8,Sigma-Aldrich)

Stringency wash composition III: 2×SSC (pH 7.0), 50% formamide(15515-026, Invitrogen)

FISH Probe Composition A: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.0.

FISH Probe Composition B: Standard sales vial of HER2PharmDx probe mix(K5331, Dako) containing blocking PNA and formamide.

Samples were denaturated at 82° C. for 5 min., hybridized at 45° for 1 hfor FISH Probe Composition A and overnight (20 hours) for FISH ProbeComposition B. All stringency washes were performed at 42° C. for 15 minfollowed by a wash in 2×SCC at 42° C. for 3×5 min.

Results:

Signal Intensity Stringency Wash Probe buffer Background DNA PNAReference (I) EC (A)   +3.0 1.5 2.5 EC (II) EC (A) +2 2.5 2.5 Formamide(III) EC (A) +1½  2.5 2.5 Reference (I) Formamide (B) +1 2.5 2.5 EC (II)Formamide (B) +½ 3 3 Formamide (III) Formamide (B) +½ 3 2.5

These results show that the buffers of the invention produce reducedbackground staining compared with 2×SSC wash buffer (Stringency washcomposition I). These results also show that the buffers of theinvention can be used instead of formamide-containing buffers forstringency washes at 42° C.

Example 24

This example compares the background and signal intensity as a functionof the stringency wash conditions. The reference was done as describedin the methods of the “general application”.

This example used a standard sales vial of HER2PharmDx probe mix (K5331,Dako) containing 10 ng/μL HER2 TxRed labeled DNA probe and (600 nM) ofCEN17 PNA probes, 45% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mMPhosphate buffer, 5 μM unlabelled blocking PNAs.

Stringency wash composition I: 2×SSC (pH 7.0), 20% EC (E2, 625-8,Sigma-Aldrich)

Stringency wash composition 2×SSC (pH 7.0), 50% EC (E2, 625-8,Sigma-Aldrich)

Stringency wash composition III: 1× Stringency Buffer (vial 4, K5599,Dako)

The samples were denatured at 82° C. for 5 min. and hybridized overnight(20 hours). The stringency wash for compositions I and II was performedat 45° C. for 15 min. followed by a wash in 2×SCC at 45° C. for 3×5 min.The stringent wash for composition III was performed at 65° C. for 10min. followed by a wash for 2×3 min. in Wash buffer (Vial 3, K5599).

Results:

Signal Intensity Stringency Wash Temperature Background DNA PNA 20% EC(I) 45° C./15 min +½ 2½ 2½ 50% EC (II) 45° C./15 min +½ 2½ 2½ Reference(III) 65° C./10 min +½ 2½ 2½

These results show that at 45° C., the buffers of the invention producesimilar levels of background as traditional stringent wash buffers at65° C. Thus, the buffers of the invention can be used to produce lowbackground under low temperature stringency wash conditions.

Example 25

This example compares the background and signal intensity as a functionof the stringency wash conditions.

This example used a standard sales vial of HER2PharmDx probe mix (K5331,Dako) containing 10 ng/μL HER2 TxRed labeled DNA probe and (600 nM) ofCEN17 PNA probes, 45% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mMPhosphate buffer, 5 μM unlabelled blocking PNAs.

Stringency wash composition I: 2×SSC (pH 7.0), 20% SL

Stringency wash composition II: 2×SSC (pH 7.0), 20% GBL

Stringency wash composition III: 1× Stringency Buffer vial 4, K5599,Dako)

The samples were denatured at 82° C. for 5 min. and hybridized overnight(20 hours). The stringency wash for compositions I and II was performedat 45° C. for 15 min. followed by a wash in 2×SCC at 45° C. for 3×5 min.The stringency wash for composition III was performed at 65° C. for 10min. followed by a wash for 2×3 min. in Wash buffer (Vial 3, K5599).

Results:

Signal Intensity Stringency Wash Temperature Background DNA PNA SL (I)45° C./15 min +0 1½-2 2½ GBL (II) 45° C./15 min +0-½ 2½-3 2½ Reference(III) 65° C./10 min +0-½    2-2½ 2½

These results show that at 45° C., the buffers of the invention producesimilar levels of background as traditional stringent wash buffers at65° C. Thus, the buffers of the invention can be used to produce lowbackground under low temperature stringency wash conditions.

Example 26

This example compares the background and signal intensity as a functionof the stringency wash conditions.

This example used a standard sales vial of HER2PharmDx probe mix (K5331,Dako) containing 10 ng/μL HER2 TxRed labeled DNA probe and (600 nM) ofCEN17 PNA probes, 45% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mMPhosphate buffer, 5 μM unlabelled blocking PNAs.

Stringency wash composition I: 0.1×SSC (pH 7.0), 20% EC

Stringency wash composition II, 0.5×SSC (pH 7.0), 20% EC

Stringency wash composition III: 1×SSC (pH 7.0), 20% EC

Stringency wash composition IV: 1× Stringency Buffer vial 4, K5599(Dako)

The samples were denatured at 82° C. for 5 min. and hybridized overnight(20 hours). The stringency wash for compositions I-III was performed at45° C. for 15 min followed by a wash in 2×SCC at 45° C. for 3×5 min,then washed 1×3 min. in Wash buffer (Vial 3, K5599). The stringency washfor composition IV was performed at 65° C. for 10 min. followed by awash for 2×3 min. in Wash buffer (Vial 3, K5599).

Results:

Signal Intensity Stringency Wash Temperature Background DNA PNA 0.1xSSC(I) 45° C./15 min +1 2½-3 2½-3 0.5xSSC (II) 45° C./15 min +1   2-3 2½-31xSSC (III) 45° C./15 min +0-2 2½-3 2½-3 Reference (IV) 65° C./10 min +12½-3 2½

These results show that at 45° C., the buffers of the invention producesimilar levels of background as traditional stringent wash buffers at65° C. Thus, the buffers of the invention can be used to produce lowbackground under low temperature stringency wash conditions withdifferent salt concentrations.

FURTHER EMBODIMENTS Embodiment 1

A kit for performing a hybridization application comprising:

-   -   at least one nucleic acid sequence; and    -   an aqueous composition for performing a stringent wash, said        composition comprising at least one polar aprotic solvent in an        amount effective to denature non-complementary sequences in a        hybridization product, and a hybridization solution,        wherein the polar aprotic solvent is not dimethyl sulfoxide        (DMSO).

Embodiment 2

The kit according to embodiment 1, wherein the concentration of polaraprotic solvent in the aqueous composition is about 1% to 95% (v/v)

Embodiment 3

The kit according to embodiment 1 or 2, wherein the concentration ofpolar aprotic solvent in the aqueous composition is 5% to 10% (v/v).

Embodiment 4

The kit according to embodiment 1 or 2, wherein the concentration ofpolar aprotic solvent in the aqueous composition is 10% to 20% (v/v).

Embodiment 5

The kit according to embodiment 1 or 2, wherein the concentration ofpolar aprotic solvent in the aqueous composition is 30% to 95% (v/v).

Embodiment 6

The kit according to any one of embodiments 1 to 5, wherein the aqueouscomposition is non-toxic.

Embodiment 7

The kit according to any one of embodiments 1 to 6, with the provisothat the aqueous composition does not contain formamide.

Embodiment 8

The kit according to embodiment 6, with the proviso that the aqueouscomposition contains less than 25% formamide.

Embodiment 9

The kit according to embodiment 8, with the proviso that the aqueouscomposition contains less than 10% formamide.

Embodiment 10

The kit according to embodiment 9, with the proviso that the aqueouscomposition contains less than 2% formamide.

Embodiment 11

The kit according to embodiment 10, with the proviso that the aqueouscomposition contains less than 1% formamide.

Embodiment 12

The kit according to any of embodiments 1 to 11, wherein the polaraprotic solvent has lactone, sulfone, nitrile, sulfite, and/or carbonatefunctionality.

Embodiment 13

The kit according to any one of embodiments 1 to 12, wherein the polaraprotic solvent has a dispersion solubility parameter between 17.7 to22.0 MPa^(1/2), a polar solubility parameter between 13 to 23 MPa^(1/2),and a hydrogen bonding solubility parameter between 3 to 13 MPa^(1/2).

Embodiment 14

The kit according to any one of embodiments 1 to 13, wherein the polaraprotic solvent has a cyclic base structure.

Embodiment 15

The kit according to any one of embodiments 1 to 13, wherein the polaraprotic solvent is selected from the group consisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S,where Z is optional and if present, is chosen from O or S,where A and B independently are O or N or S or part of the alkyldiyl ora primary amine,where R is alkyldiyl, andwhere Y is O or S or C.

Embodiment 16

The kit according to any one of embodiments 1 to 15, wherein the polaraprotic solvent is selected from the group consisting of: acetanilide,acetonitrile, N-acetyl pyrrolidone, 4-amino pyridine, benzamide,benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylenecarbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleicanhydride, 2-chlorocyclohexanone, chloroethylene carbonate,chloronitromethane, citraconic anhydride, crotonlactone,5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethylsulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate,N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate,ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile,2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile,1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methylimidazole, N-methyl imidazole, 3-methyl isoxazole, N-methylmorpholine-N-oxide, methyl phenyl sulfone, N-methylpyrrolidinone, methylsulfolane, methyl-4-toluenesulfonate, 3-nitroaniline,nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrrolidinone,2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine),1,3-propane sultone, β-propiolactone, propylene carbonate,4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone,saccharin, succinonitrile, sulfanilamide, sulfolane,2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, and trimethylene sulfite.

Embodiment 17

The kit according to any one of embodiments 1 to 15, wherein the polaraprotic solvent is selected from the group consisting of:

Embodiment 18

The kit according to any one of embodiments 1 to 15, wherein the polaraprotic solvent is:

Embodiment 19

The kit according to any one of embodiments 1 to 18, wherein the aqueouscomposition further comprises at least one additional component selectedfrom the group consisting of: buffering agents, salts, acceleratingagents, chelating agents, and detergents.

Embodiment 20

The kit according to embodiment 19, wherein the salts are NaCl and/orphosphate buffer and/or citrate buffer.

Embodiment 21

The kit according to embodiment 19, wherein the salts are NaCl and/orphosphate buffer and/or SSC.

Embodiment 22

The kit according to embodiment 20, wherein the NaCl is present at aconcentration of 0 mM to 1200 mM and/or the citrate buffer is present ata concentration of 0 mM to 100 mM.

Embodiment 23

The kit according to embodiment 22, wherein the NaCl is present at aconcentration of 50 mM to 600 mM and/or the citrate buffer is present ata concentration of 5 mM to 50 mM.

Embodiment 24

The kit according to embodiment 19, wherein the accelerating agent isselected from the group consisting of: formamide, DMSO, glycerol,propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol,glycol, 1,2 propanediol, and 1,3 propanediol.

Embodiment 25

The kit according to embodiment 24, wherein the formamide is present ata concentration of 0.1-5%, the DMSO is present at a concentration of0.01% to 10%, the glycerol, propylene glycol, 1,2-propanediol,diethylene glycol, ethylene glycol, glycol, 1,2 propanediol, and 1,3propanediol are present at a concentration of 0.1% to 10%, and thecitric acid buffer is present at a concentration of 1 mM to 100 mM.

Embodiment 26

The kit according to any one of embodiments 1-25, wherein the aqueouscomposition comprises 50% of at least one polar aprotic solvent and0.05× to 4×SSC.

Embodiment 27

The kit according to any one of embodiments 1-25, wherein the aqueouscomposition comprises 20% of at least one polar aprotic solvent and2×SSC.

Embodiment 28

The kit according to any one of embodiments 1-25, wherein the aqueouscomposition comprises 15% of at least one polar aprotic solvent and2×SSC.

Embodiment 29

The kit according to any one of embodiments 1-28, wherein the aqueouscomposition comprises more than one phase at room temperature.

Embodiment 30

The kit according to embodiment 29, wherein the aqueous compositioncomprises two phases at room temperature.

Embodiment 31

The kit according to embodiment 29, wherein the aqueous compositioncomprises three phases at room temperature.

Embodiment 32

A method for performing a stringent wash step in a hybridizationapplication comprising:

-   -   a. providing a hybridization product comprising a first nucleic        acid sequence hybridized to a second nucleic acid sequence,    -   b. providing an aqueous composition comprising at least one        polar aprotic solvent in an amount effective to denature        non-complementary double-stranded nucleotide sequences, and    -   c. combining the hybridization product and the aqueous        composition for at least a time period sufficient to denature        any non-complementary binding between the first and second        nucleic acid sequences.        wherein the polar aprotic solvent is not dimethyl sulfoxide        (DMSO).

Embodiment 33

A method for performing a stringent wash step in a hybridizationapplication comprising:

-   -   a. providing a hybridization product comprising a first nucleic        acid sequence hybridized to a second nucleic acid sequence,    -   b. providing an aqueous composition as defined in any of        embodiments 1 to 31, and    -   c. combining the hybridization product and the aqueous        composition for at least a time period sufficient to denature        any non-complementary binding between the first and second        nucleic acid sequences.

Embodiment 34

The method according to embodiment 32, wherein the polar aprotic solventis defined according to any of embodiments 2-5 or 12-18.

Embodiment 35

The method according to embodiment 32 or 33, wherein a sufficient amountof energy to denature any non-complementary binding between the firstand second nucleic acid sequences is provided.

Embodiment 36

The method according to embodiment 35, wherein the energy is provided byheating the combination produced in (c).

Embodiment 37

The method according to embodiment 36, wherein the energy is provided bythe use of microwaves, hot baths, hot plates, heat wire, peltierelement, induction heating, or heat lamps.

Embodiment 38

The method according to embodiment 36, wherein the combination producedin (c) is heated to less than 70° C., such as, for example, 65° C., 62°C., 60° C., 55° C., 52° C., 50° C., 45° C., 42° C., 40° C., 35° C., 30°C., or room temperature.

Embodiment 39

The method according to any of embodiments 32 to 38, wherein thedenaturation of any non-complementary binding between the first andsecond nucleic acid sequences occurs in less than 1 hour, such as, forexample, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1minute.

Embodiment 40

The method according to any one of embodiments 32-39, wherein the firstnucleic acid sequence is in a biological sample.

Embodiment 41

The method according to embodiment 40, wherein the biological sample isa cytology or histology sample.

Embodiment 43

The method according to any one of embodiments 32-41, wherein theaqueous composition comprises one phase at room temperature.

Embodiment 44

The method according to any one of embodiments 32-43, wherein theaqueous composition comprises multiple phases at room temperature.

Embodiment 45

The method according to embodiment 44, wherein the aqueous compositioncomprises two phases at room temperature.

Embodiment 46

The method according to embodiment 44 or 45, wherein the phases of theaqueous composition are mixed.

Embodiment 47

An aqueous composition for performing a stringent wash in ahybridization application, said composition comprising at least onepolar aprotic solvent in an amount effective to denaturenon-complementary sequences in a hybridization product, and ahybridization solution, wherein the polar aprotic solvent is notdimethyl sulfoxide (DMSO).

Embodiment 48

The aqueous composition of embodiment 47, wherein the concentration ofpolar aprotic solvent is defined as in any one of embodiments 2 to 5.

Embodiment 49

The aqueous composition of embodiment 47 or 48, wherein the polaraprotic solvent is defined as in any one of embodiments 12 to 18.

Embodiment 50

The aqueous composition of any one of embodiments 47 to 49, wherein theaqueous composition is further defined as in any one of embodiments 6 to11 or 19 to 31.

Embodiment 51

Use of an aqueous composition comprising between 1 and 95% (v/v) of atleast one polar aprotic solvent for a stringent wash step in ahybridization application.

Embodiment 52

Use of a composition according to embodiment 51, wherein the aqueouscomposition is defined as in any one of embodiments 1 to 31.

1. A kit for performing a hybridization application comprising: at leastone nucleic acid sequence; and an aqueous composition for performing astringent wash, said composition comprising at least one polar aproticsolvent in an amount effective to denature non-complementary sequencesin a hybridization product, and a hybridization solution, wherein thepolar aprotic solvent is not dimethyl sulfoxide (DMSO).
 2. The kitaccording to claim 1, wherein the concentration of polar aprotic solventin the aqueous composition is about 1% to 95% (v/v)
 3. The kit accordingto claim 2, wherein the concentration of polar aprotic solvent in theaqueous composition is 5% to 10% (v/v).
 4. The kit according to claim 2,wherein the concentration of polar aprotic solvent in the aqueouscomposition is 10% to 20% (v/v).
 5. The kit according to claim 2,wherein the concentration of polar aprotic solvent in the aqueouscomposition is 30% to 95% (v/v).
 6. The kit according to claim 1,wherein the aqueous composition is non-toxic.
 7. The kit according toclaim 1, with the proviso that the aqueous composition does not containformamide.
 8. The kit according to claim 6, with the proviso that theaqueous composition contains less than 25% formamide.
 9. The kitaccording to claim 8, with the proviso that the aqueous compositioncontains less than 10% formamide.
 10. The kit according to claim 9, withthe proviso that the aqueous composition contains less than 2%formamide.
 11. The kit according to claim 10, with the proviso that theaqueous composition contains less than 1% formamide.
 12. The kitaccording to claim 1, wherein the polar aprotic solvent has lactone,sulfone, nitrile, sulfite, and/or carbonate functionality.
 13. The kitaccording to claim 1, wherein the polar aprotic solvent has a dispersionsolubility parameter between 17.7 to 22.0 MPa^(1/2), a polar solubilityparameter between 13 to 23 MPa^(1/2), and a hydrogen bonding solubilityparameter between 3 to 13 MPa^(1/2).
 14. The kit according to claim 1,wherein the polar aprotic solvent has a cyclic base structure.
 15. Thekit according to claim 1, wherein the polar aprotic solvent is selectedfrom the group consisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S, where Z isoptional and if present, is chosen from O or S, where A and Bindependently are O or N or S or part of the alkyldiyl or a primaryamine, where R is alkyldiyl, and where Y is O or S or C.
 16. The kitaccording to claim 1, wherein the polar aprotic solvent is selected fromthe group consisting of: acetanilide, acetonitrile, N-acetylpyrrolidone, 4-amino pyridine, benzamide, benzimidazole,1,2,3-benzotriazole, butadienedioxide, 2,3-butylene carbonate,γ-butyrolactone, caprolactone (epsilon), chloro maleic anhydride,2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane,citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil,cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone,1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole, 1,2-dinitrobenzene,2,4-dinitrotoluene, dipheynyl sulfone, 1,2-dinitrobenzene,2,4-dinitrotoluene, dipheynyl sulfone, epsilon-caprolactam,ethanesulfonylchloride, ethyl ethyl phosphinate, N-ethyl tetrazole,ethylene carbonate, ethylene trithiocarbonate, ethylene glycol sulfate,glycol sulfite, furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole,malononitrile, 4-methoxy benzonitrile, 1-methoxy-2-nitrobenzene, methylalpha bromo tetronate, 1-methyl imidazole, N-methyl imidazole, 3-methylisoxazole, N-methyl morpholine-N-oxide, methyl phenyl sulfone, N-methylpyrrolidinone, methyl sulfolane, methyl-4-toluenesulfonate,3-nitroaniline, nitrobenzimidazole, 2-nitrofuran,1-nitroso-2-pyrrolidinone, 2-nitrothiophene, 2-oxazolidinone,9,10-phenanthrenequinone, N-phenyl sydnone, phthalic anhydride,picolinonitrile (2-cyanopyridine), 1,3-propane sultone, β-propiolactone,propylene carbonate, 4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone),pyridazine, 2-pyrrolidone, saccharin, succinonitrile, sulfanilamide,sulfolane, 2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, and trimethylene sulfite.
 17. Thekit according to claim 1, wherein the polar aprotic solvent is selectedfrom the group consisting of:


18. The kit according to claim 1, wherein the polar aprotic solvent is:


19. The kit according to claim 1, wherein the aqueous compositionfurther comprises at least one additional component selected from thegroup consisting of: buffering agents, salts, accelerating agents,chelating agents, and detergents.
 20. The kit according to claim 19,wherein the salts are NaCl and/or phosphate buffer and/or citratebuffer.
 21. The kit according to claim 19, wherein the salts are NaCland/or phosphate buffer and/or SSC.
 22. The kit according to claim 20,wherein the NaCl is present at a concentration of 0 mM to 1200 mM and/orthe citrate buffer is present at a concentration of 0 mM to 100 mM. 23.The kit according to claim 22, wherein the NaCl is present at aconcentration of 50 mM to 600 mM and/or the citrate buffer is present ata concentration of 5 mM to 50 mM.
 24. The kit according to claim 19,wherein the accelerating agent is selected from the group consisting of:formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethyleneglycol, ethylene glycol, glycol, 1,2 propanediol, and 1,3 propanediol.25. The kit according to claim 24, wherein the formamide is present at aconcentration of 0.1-5%, the DMSO is present at a concentration of 0.01%to 10%, the glycerol, propylene glycol, 1,2-propanediol, diethyleneglycol, ethylene glycol, glycol, 1,2 propanediol, and 1,3 propanediolare present at a concentration of 0.1% to 10%, and the citric acidbuffer is present at a concentration of 1 mM to 100 mM.
 26. The kitaccording to claim 1, wherein the aqueous composition comprises 50% ofat least one polar aprotic solvent and 0.05× to 4×SSC.
 27. The kitaccording to claim 1, wherein the aqueous composition comprises 20% ofat least one polar aprotic solvent and 2×SSC.
 28. The kit according toclaim 1, wherein the aqueous composition comprises 15% of at least onepolar aprotic solvent and 2×SSC.
 29. The kit according to claim 1,wherein the aqueous composition comprises more than one phase at roomtemperature.
 30. The kit according to claim 29, wherein the aqueouscomposition comprises two phases at room temperature.
 31. The kitaccording to claim 29, wherein the aqueous composition comprises threephases at room temperature.
 32. (canceled)
 33. A method for performing astringent wash step in a hybridization application comprising: a.providing a hybridization product comprising a first nucleic acidsequence hybridized to a second nucleic acid sequence, b. providing anaqueous composition as defined in claim 1, and c. combining thehybridization product and the aqueous composition for at least a timeperiod sufficient to denature any non-complementary binding between thefirst and second nucleic acid sequences.
 34. (canceled)
 35. The methodaccording to claim 33, wherein a sufficient amount of energy to denatureany non-complementary binding between the first and second nucleic acidsequences is provided.
 36. The method according to claim 35, wherein theenergy is provided by heating the combination produced in (c).
 37. Themethod according to claim 36, wherein the energy is provided by the useof microwaves, hot baths, hot plates, heat wire, peltier element,induction heating, or heat lamps.
 38. The method according to claim 36,wherein the combination produced in (c) is heated to less than 70° C.,such as, for example, 65° C., 62° C., 60° C., 55° C., 52° C., 50° C.,45° C., 42° C., 40° C., 35° C., 30° C., or room temperature.
 39. Themethod according to claim 33, wherein the denaturation of anynon-complementary binding between the first and second nucleic acidsequences occurs in less than 1 hour, such as, for example, 45 minutes,30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.
 40. Themethod according to claim 33, wherein the first nucleic acid sequence isin a biological sample.
 41. The method according to claim 40, whereinthe biological sample is a cytology or histology sample.
 42. The methodaccording to claim 33, wherein the aqueous composition comprises onephase at room temperature.
 43. The method according to claim 33, whereinthe aqueous composition comprises multiple phases at room temperature.44. The method according to claim 43, wherein the aqueous compositioncomprises two phases at room temperature.
 45. The method according toclaim 43, wherein the phases of the aqueous composition are mixed. 46.An aqueous composition for performing a stringent wash in ahybridization application, said composition comprising at least onepolar aprotic solvent in an amount effective to denaturenon-complementary sequences in a hybridization product, and ahybridization solution, wherein the polar aprotic solvent is notdimethyl sulfoxide (DMSO). 47-51. (canceled)